JP2016172779A - Influenza vaccine, composition, and methods of use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vaccine and composition that induces an immune response against one or more influenza strains using a mutated Bordetella strain capable of causing pertussis as an active principle agent.SOLUTION: A method for inducing an immune response against influenza viruses in a mammal comprises a step of administering a mutated Bordetella strain, to a mammal, which comprises a mutated pertussis toxin (ptx) gene, a deleted or mutated dermonecrotic (dnt) gene, and a heterologous ampG gene. The mutated Bordetella strain is a pharmacological composition in which a wild-type Bordetella strain ampG gene is replaced by an E. coli ampG gene.SELECTED DRAWING: Figure 4

Description

Field The present invention relates to the fields of microbiology and virology.

background
There are three types of influenza viruses: type A, type B and type C, and their epidemiological patterns are very different. Influenza A virus is the best characterized virus and is also the most serious wonder to public health with the ability to cause large-scale or pandemic. This virus is also very diverse antigenically making it difficult to produce an effective vaccine.

  Vaccines against influenza are one of the most effective, safe, non-toxic and economical weapons for preventing disease and controlling disease spread. The primary purpose of vaccination is to activate specific adaptive immune responses, primarily to generate B and T lymphocytes against antigens associated with the disease or disease factor.

  Currently, several vaccines against influenza are available and mainly consist of inactivated vaccines. These vaccines can include two types of A antigens (eg, H1N1 and H3N2) and one type B antigen. Available vaccines typically include whole virion, degradation products, and subunit vaccines. In general, these vaccines are effective in up to 90% of vaccinated individuals when the vaccine closely matches the identity of emerging epidemics. However, they need to be updated every year to accommodate the influenza virus antigenic variation.

  Furthermore, a live intranasal attenuated vaccine (LAIV) adapted to low temperatures against seasonal influenza has recently been described. Cross-protective immunity has been demonstrated in studies reporting protection against H3N2 virus in cotton rats infected with the H1N1 strain. However, one of the major concerns associated with LAIV is the possibility of genetic reversion and gene reassortment with wild-type influenza viruses, resulting in new latently infected strains.

  The present invention addresses these and other issues in the influenza vaccine area by providing vaccines and compositions that elicit an immune response against one or more influenza strains, using a mutant Bordetella strain as the active agent. To address the problem.

  In addition, the related art describes various types of vaccines and compositions using Bordetella strains, for example, to induce an immune response against Bordetella bacteria that have the ability to cause whooping cough in humans (WO2007104451 (Patents)). 1) and WO2003102170) describe a method or composition for inducing an immune response to an influenza virus in a mammal using the method, composition and / or vaccine of the present invention. Not disclosed.

  Thus, there is a need for new influenza vaccines that can provide broad protection against diseases caused by influenza virus infection.

WO2007104451 WO2003102170

SUMMARY The present invention relates to compositions and vaccines comprising mutant Bordetella strains for treating or preventing influenza infection in mammals. In addition, the present invention further provides methods for protecting a mammal against infection with influenza and / or for inducing an immune response against an influenza virus in a mammal using the compositions and vaccines.

  In one aspect, the present invention provides a method for inducing an immune response against an influenza virus in a mammal comprising administering a mutant Bordetella strain to the mammal, wherein the strain comprises a mutated pertussis toxin (ptx) gene, Methods are provided comprising a deleted or mutated skin necrosis (dnt) gene and a heterologous ampG gene. In some aspects, the Bordetella strain comprises a Bordetella pertussis strain. In some such aspects, the wild type Bordetella strain ampG gene has been replaced by the E. coli ampG gene. In other aspects, mutations in the ptx gene include substitution of amino acids involved in substrate binding and / or amino acids involved in catalysis. In some such aspects, substitutions of amino acids involved in substrate binding include K9R and substitutions of amino acids involved in catalysis include E129G. In some aspects, the Bordetella strain comprises a triple mutant strain. In some such aspects, the Bordetella strain comprises the BPZE1 strain. In other such aspects, the Bordetella strain is attenuated. In some aspects, the Bordetella strain comprises a live strain. In other aspects, the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene. In some aspects, the Bordetella strain does not include a heterologous expression platform for delivering a heterologous antigen to the mammalian respiratory mucosa. In other aspects, the method further comprises preventing or treating influenza infection in the mammal. In some aspects, the Bordetella strain is administered prior to influenza infection. In some such aspects, the Bordetella strain is administered about 6 weeks or prior to influenza infection. In other such aspects, the Bordetella strain is administered about 12 weeks or prior to influenza infection. In some aspects, the influenza virus comprises H3 or H1. In other aspects, the influenza virus comprises N2 or N1.

  In some aspects, the influenza virus comprises H3 and N2. In other aspects, the influenza virus comprises H1 and N1. In some aspects, the immune response comprises a Th1 immune response. In some aspects, the strain is administered subcutaneously (sc), intradermally (id), intramuscularly (im), intravenously (iv), orally or intranasally, or by infusion, or by inhalation To be administered to a mammal. In other aspects, the strain is administered intranasally. In some other aspects, the strain is administered to a mammal in need of protective immunity against influenza infection. In some aspects, the mammal is a child. In some aspects, the strain is administered once. In some aspects, the strain is administered more than once. In some such aspects, the strain is administered twice. In other aspects, it is administered twice, about 3 weeks apart. In some aspects, the level of protection against influenza infection is greater than about 60%. In other aspects, the level of protection against influenza infection is greater than about 50%. In some aspects, the mammal is a human.

  In another aspect, the present invention elicits a protective immune response against H3N2 influenza virus in humans, comprising intranasally administering a human live attenuated BPZE1 strain prior to human infection with H3N2 influenza virus A method wherein the strain does not include a heterologous expression platform for delivering a heterologous antigen to the human respiratory mucosa.

  In another aspect, the present invention provides a method for inducing an immune response against influenza virus in a human comprising administering a live Bordetella strain to the human, wherein the strain carries a heterologous antigen to the human respiratory mucosa. Methods are provided that do not include a heterologous expression platform.

  In another aspect, the present invention is directed against a disease caused by influenza infection comprising administering to a mammal a mutated Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene. A method of protecting a mammal is provided.

  In another aspect, the present invention provides protective immunity against influenza infection comprising administering to a mammal a mutated Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene.

  In another aspect, the invention provides a composition for treating or preventing influenza infection in a mammal, including a mutated Bordetella strain, wherein the strain is mutated pertussis toxin (ptx) gene, deleted or mutated Compositions comprising a skin necrosis (dnt) gene and a heterologous ampG gene are provided. In some aspects, the Bordetella strain comprises a Bordetella pertussis strain. In other aspects, the wild type Bordetella strain ampG gene is replaced with the E. coli ampG gene. In some aspects, mutations in the ptx gene include substitution of amino acids involved in substrate binding and / or amino acids involved in catalysis. In other aspects, substitutions of amino acids involved in substrate binding include K9R and substitutions of amino acids involved in catalysis include E129G. In some aspects, the Bordetella strain comprises a triple mutant strain.

  In another aspect, the present invention provides a vaccine comprising a composition of the present invention for treating or preventing influenza infection in a mammal. In some aspects, the vaccine comprises a Bordetella strain composition described herein. In some aspects, the vaccine is formulated for intranasal administration.

  In another aspect, the present invention provides a Bordetella strain identified by accession number CNCM I-3585.

In another aspect, the invention provides a Bordetella strain identified by Accession No. V09 / 009169.
[Invention 1001]
A method of inducing an immune response against an influenza virus in a mammal comprising administering a mutant Bordetella strain to the mammal, wherein the strain comprises a mutated pertussis toxin (ptx) gene, a deficiency A method comprising a lost or mutated skin necrosis (dnt) gene and a heterologous ampG gene.
[Invention 1002]
The method of the present invention 1001, wherein the Bordetella strain comprises a Bordetella pertussis strain.
[Invention 1003]
The method of the present invention 1002, wherein the wild-type Bordetella strain ampG gene is replaced by an E. coli ampG gene.
[Invention 1004]
The method of the present invention 1003, wherein the mutation in the ptx gene comprises a substitution of an amino acid involved in substrate binding and / or an amino acid involved in catalysis.
[Invention 1005]
The method of 1004 of this invention, wherein the substitution of amino acids involved in substrate binding comprises K9R and the substitution of amino acids involved in catalysis comprises E129G.
[Invention 1006]
The method of the present invention 1001, wherein the Bordetella strain comprises a triple mutant strain.
[Invention 1007]
The method of the present invention 1006, wherein the Bordetella strain comprises the BPZE1 strain.
[Invention 1008]
The method of the present invention 1001, wherein the Bordetella strain is attenuated.
[Invention 1009]
The method of 1001 of the present invention, wherein the Bordetella strain comprises a live strain.
[Invention 1010]
The method of the present invention 1001, wherein the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene.
[Invention 1011]
The method of the present invention 1001, wherein the Bordetella strain does not include a xenogeneic expression platform for delivering xenoantigens to the mammalian respiratory mucosa.
[Invention 1012]
The method of the present invention 1001, further comprising the step of preventing or treating influenza infection in a mammal.
[Invention 1013]
The method of the present invention 1001, wherein the Bordetella strain is administered prior to influenza infection.
[Invention 1014]
The method of the present invention 1013 wherein the Bordetella strain is administered about 6 weeks or prior to influenza infection.
[Invention 1015]
The method of the present invention 1014 wherein the Bordetella strain is administered about 12 weeks or prior to influenza infection.
[Invention 1016]
The method of 1001 of this invention, wherein the influenza virus comprises H3 or H1.
[Invention 1017]
The method of the present invention 1001, wherein the influenza virus comprises N2 or N1.
[Invention 1018]
The method of the present invention 1001, wherein the influenza virus comprises H3 and N2.
[Invention 1019]
The method of the present invention 1001, wherein the influenza virus comprises H1 and N1.
[Invention 1020]
The method of 1001 of this invention wherein the immune response comprises a Th1 immune response.
[Invention 1021]
The invention wherein the strain is administered to a mammal by subcutaneous (sc), intradermal (id), intramuscular (im), intravenous (iv), oral, or intranasal administration; or by infusion or by inhalation 1001 way.
[Invention 1022]
The method of the present invention 1001, wherein the strain is administered intranasally.
[Invention 1023]
The method of 1001 of the present invention, wherein the strain is administered to a mammal in need of protective immunity against influenza infection.
[Invention 1024]
The method of the present invention 1001, wherein the mammal is a child.
[Invention 1025]
The method of 1001 of the present invention, wherein the strain is administered once.
[Invention 1026]
The method of 1001 of this invention, wherein the strain is administered more than once.
[Invention 1027]
The method of the present invention 1026 wherein the strain is administered twice.
[Invention 1028]
The method of the present invention 1027, which is administered twice, about 3 weeks apart.
[Invention 1029]
The method of the present invention 1018 wherein the level of protection against influenza infection is greater than about 60%.
[Invention 1030]
The method of the present invention 1019, wherein the level of protection against influenza infection is greater than about 50%.
[Invention 1031]
The method of the present invention 1001 wherein the mammal is a human.
[Invention 1032]
A method of inducing a protective immune response against a H3N2 influenza virus in a human comprising the step of intranasally administering to said human a live attenuated BPZE1 strain prior to infection of the human with the H3N2 influenza virus, wherein the strain comprises A method that does not include a heterologous expression platform for delivering a heterologous antigen to the human respiratory mucosa.
[Invention 1033]
A method of inducing an immune response against an influenza virus in a human, the method comprising administering a live Bordetella strain to the human, wherein the strain carries a heterologous antigen to the human respiratory mucosa Not including the way.
[Invention 1034]
A method for protecting a mammal against diseases caused by influenza infection, comprising the step of administering to the mammal a mutant Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene Including.
[Invention 1035]
A method of providing protective immunity against influenza infection comprising administering to a mammal a mutant Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene.
[Invention 1036]
A composition for treating or preventing influenza infection in a mammal comprising a mutated Bordetella strain comprising a mutated pertussis toxin (ptx) gene, a deleted or mutated skin necrosis (dnt) gene, and a heterologous ampG gene ,Composition.
[Invention 1037]
The composition of the invention 1036 wherein the Bordetella strain comprises a Bordetella pertussis strain.
[Invention 1038]
The composition of the invention 1037 wherein the wild type Bordetella strain ampG gene is replaced by the E. coli ampG gene.
[Invention 1039]
The composition of the invention 1038, wherein a mutation in the ptx gene comprises a substitution of an amino acid involved in substrate binding and / or an amino acid involved in catalysis.
[Invention 1040]
The composition of the invention 1039 wherein the substitution of amino acids involved in substrate binding comprises K9R and the substitution of amino acids involved in catalysis comprises E129G.
[Invention 1041]
The composition of the invention 1036 wherein the Bordetella strain comprises a triple mutant strain.
[Invention 1042]
The composition of the present invention 1041 wherein the Bordetella strain comprises the BPZE1 strain.
[Invention 1043]
The composition of this invention 1036 wherein the Bordetella strain is attenuated.
[Invention 1044]
The composition of the present invention 1036, wherein the Bordetella strain comprises a live strain.
[Invention 1045]
The composition of the present invention 1036, wherein the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene.
[Invention 1046]
The composition of the invention 1036 wherein the Bordetella strain does not comprise a xenogeneic expression platform for delivering xenoantigens to the mammalian respiratory mucosa.
[Invention 1047]
The composition of this invention 1036 further comprising a pharmaceutically suitable excipient, vehicle and / or carrier.
[Invention 1048]
The composition of this invention 1036 further comprising an adjuvant.
[Invention 1049]
The composition of this invention 1036 further comprising a small molecule capable of affecting an influenza infection.
[Invention 1050]
A composition comprising a Bordetella strain identified by accession number CNCM I-3585.
[Invention 1051]
A composition comprising a Bordetella strain identified by Accession No. V09 / 009169.
[Invention 1052]
A vaccine comprising the composition of the invention 1036 for treating or preventing influenza infection in a mammal.
[Invention 1053]
The vaccine of the present invention 1052 formulated for intranasal administration.
[Invention 1054]
A vaccine comprising the composition of the invention 1050 for treating or preventing influenza infection in a mammal.
[Invention 1055]
The vaccine of this invention 1054 formulated for intranasal administration.
[Invention 1056]
A vaccine comprising the composition of the present invention 1051 for treating or preventing influenza infection in a mammal.
[Invention 1057]
The vaccine of the present invention 1056 formulated for intranasal administration.

  These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings.

The protection rate of BPZE1-treated mice against lethal challenge with mouse-adapted H3N2 virus is shown. Adult Balb / c mice are given nasal administration of 5 x 10 ⁶ cfu of BPZE1 bacteria and fit to a lethal dose (2LD50) of mice either 3 weeks (black squares) or 6 weeks (black triangles) Attacked by H3N2 virus. Body weight changes were monitored daily and mice were euthanized when the body weight decreased more than 20% of the original body weight. Survival was compared to untreated mice (black diamonds). Ten animals were evaluated per group. Results are representative of 3 independent experiments. Shows protection rates against lethal challenge with mouse-adapted H3N2 virus in mice treated with BPZE1 dead bacteria or live BPZE1 bacteria. Adult Balb / c mice are given 5 x 10 ⁶ cfu BPZE1 live bacteria (black triangles) or BPZE1 dead bacteria (black squares) nasally and challenged with a lethal dose (2LD50) of mouse-matched H3N2 virus 6 weeks later did. Body weight changes were monitored daily and mice were euthanized when the body weight decreased more than 20% of the original body weight. Survival was compared to untreated mice (black diamonds). Ten animals were evaluated per group. Results are representative of two independent trials. Figure 3 shows the protection rate against lethal challenge with mouse-adapted H3N2 virus in mice treated twice with live BPZE1 bacteria. Adult Balb / c mice were given two nasal doses of 5 × 10 ⁶ cfu of BPZE1 bacteria (black squares) at 4-week intervals and 4 weeks later challenged with a lethal dose (2LD50) of mouse-matched H3N2 virus . Body weight changes were monitored daily and mice were euthanized when the body weight decreased more than 20% of the original body weight. Survival was compared to untreated mice (black diamonds). Ten animals were evaluated per group. Results are representative of two independent experiments. The protection rate of BPZE1-treated mice against lethal challenge with H1N1 influenza A virus is shown. Adult Balb / c mice were given 5 × 10 ⁶ cfu viable BPZE1 bacteria 3 times at 4 and 3 week intervals (black triangles). Animals were challenged with a lethal dose (4LD50) of A / PR / 8/34 (H1N1) influenza A virus 2 weeks after the last BPZE1 treatment. Body weight changes were monitored daily and mice were euthanized when the body weight decreased more than 20% of the original body weight. Survival was compared to untreated mice (black diamonds). Ten animals were evaluated per group. Results are representative of two independent experiments. Figure 2 shows viral load quantification in the lungs of protected versus unprotected mice. Adult Balb / c mice were given 5 × 10 ⁶ cfu viable BPZE1 bacteria nasally twice at 4-week intervals and challenged 4 weeks later with a lethal dose (2LD50) of mouse-matched H3N2 virus. Five animals per group were sacrificed 3 days after virus challenge and their lungs were collected and individually processed for in vitro determination of TCID ₅₀ for infection with MDCK cells. Viral load was compared to that obtained in untreated mice. Results are representative of 3 independent experiments. Shown are pulmonary histology and cell infiltrates and CD3 ⁺ T cell populations in the lungs of BPZE1-treated versus untreated mice after lethal virus challenge. Adult Balb / c mice were administered nasally with 5 × 10 ⁶ cfu of BPZE1 live bacteria and 6 weeks later challenged with a lethal dose (2LD50) of mouse-matched H3N2 virus. Three days after viral challenge, mice were euthanized and their lungs were individually processed for histological analysis (A) or bronchoalveolar lavage for cell infiltrate analysis (B). Legend for A: Infected control mice exhibited severe inflammation, pulmonary edema (black arrow) and severe necrotizing bronchitis due to necrotic cell debris (white arrow). Infected BPZE1-treated mice showed only minimal inflammation and airway injury, as well as mild peribronchial injury. Representative areas are shown. Results were comparable in more than 40 analysis areas per group (> 5 areas / part, 2 parts / mouse and 4 mice / group). B legend: a, untreated untreated mice; b, untreated mice challenged with H3N2 virus; c, non-attacked BPZE1-treated mice; d, BPZE1-treated mice challenged with H3N2 virus. Four animals were evaluated individually at each time point per group. ^** , p ≦ 0.01; ^*** , p ≦ 0.001. Results are representative of two independent experiments. (C) FACS analysis of CD3 ⁺ T cell population in mouse lung. Three or five days after lethal H3N2 influenza virus challenge, untreated control mice and mice treated twice with BPZE1 were euthanized and the CD3 ⁺ T cell population in their lungs was analyzed by flow cytometry. Four animals were evaluated individually at each time point per group. Results are expressed as a percentage of CD3 ⁺ T cells in the total lung cell population. Mean value ± SD. Legend: a, untreated untreated mice; b, untreated BPZE1-treated mice; c, untreated mice treated with H3N2 virus and sacrificed 3 days later; d, challenged with H3N2 virus and 3 days later BPZE1-treated mice sacrificed; e, untreated mice challenged with H3N2 virus and sacrificed 5 days later; f, BPZE1-treated mice challenged with H3N2 virus and sacrificed 5 days later. ^*** , P ≦ 0.001. Figure 5 shows proinflammatory cytokines, anti-inflammatory cytokines and chemokine profiles in BPZE1-treated versus untreated mice after lethal H3N2 virus challenge. Adult Balb / c mice were given 5 × 10 ⁶ cfu viable BPZE1 bacteria nasally twice at 4-week intervals and challenged with a lethal dose (2LD50) of mouse-matched H3N2 virus 4 weeks after the last dose. One and three days after virus challenge, 5 mice per group were sacrificed at each time point and bronchoalveolar lavage fluid (BALF) was collected. Fourteen inflammation-related cytokines and chemokines were measured in each individual BALF sample. Legend: 1, untreated untreated mice; 2, BPZE1-treated non-challenged mice; 3, untreated challenged mice sacrificed one day after challenge; 4, BPZE1-treated challenged mice Sacrificed 1 day after challenge; 5; sacrificed 3 days after challenge in untreated challenged mice; 6; sacrificed 3 days after challenge in challenged mice treated with BPZE1. ^* , P ≦ 0.05; ^** , p ≦ 0.01; ^*** , p ≦ 0.001. Figure 2 shows the role of Bordetella pertussis specific immunity in cross-protection (A) Untreated (open triangle) or anti-H3N2 (filled circle) or anti-BPZE1 (filled circle) immune serum was administered ip to adult untreated Balb / c mice one day prior to lethal challenge with mouse-matched H3N2 virus (2LD50). Injected. Body weight changes were monitored daily and mice were euthanized when the body weight decreased more than 20% of the original body weight. Survival was compared to untreated mice (black triangles). Ten animals were evaluated per group. (B) and (C) Adult Balb / c mice were administered 5 × 10 ⁶ cfu viable BPZE1 bacteria nasally, animals were euthanized 6 weeks later, and their spleens were collected. Pooled splenocytes (B) or individual splenocytes (C) from 6 animals were stimulated with either BPZE1 lysate or heat-inactivated H3N2 virus particles. ³ H thymidine incorporation (B) and IFN-γ ELISPOT assay (C) were performed as follows. Representatives from both two different experiments showed similar results; mean ± SD; ^* , p ≦ 0.05, ^*** , p ≦ 0.001.

Detailed Description Introduction and Summary The present invention relates to compositions and vaccines comprising mutant Bordetella strains for treating or preventing influenza infection in mammals. In addition, the present invention further provides methods for protecting a mammal against infection with influenza and / or for inducing an immune response against an influenza virus in a mammal using the composition or vaccine.

  The terms used in the claims and specification are defined as follows unless otherwise specified.

  As used herein, the abbreviation “PTX” refers to pertussis toxin and synthesizes and secretes ADP-ribosylating toxin. PTX is composed of 5 different subunits (named S1-S5), each complex containing 2 copies of S4. The subunits are arranged in an A-B structure. The A component is enzymatically active and is formed from the S1 subunit, while the B component is the receptor binding moiety and is composed of subunits S2-S5.

  As used herein, the abbreviation “DNT” refers to pertussis skin necrosis toxin, a heat-labile toxin that can induce local lesions when injected intradermally in mice and other laboratory animals.

  As used herein, the abbreviation “TCT” refers to tracheal cytotoxin and is a virulence factor synthesized by Bordetella. TCT is a peptidoglycan fragment and has the ability to induce interleukin 1 production and nitric oxide synthase. It has the ability to cause cilia quiescence and has a lethal effect on respiratory epithelial cells.

  The term “attenuated” refers to a weak and less pathogenic Bordetella strain that is capable of stimulating an immune response and causing protective immunity, but generally does not cause disease.

  The term “rapid protective immunity” means that immunity against Bordetella is conferred in a short time after administration of the mutant Bordetella strain of the invention.

  The term “bordetella strain” or “strain” includes strains derived from Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.

  The term “child” is intended to be a human or mammal between 0 months and 18 years old.

  `` Treat '' means abatement, remission, symptomatic relief or a condition that is acceptable to the patient, slowing down the rate of degeneration or decay, or debilitating the end point of degeneration Refers to any sign of successful treatment or amelioration or prevention of a disease, condition or disorder, including any subjective or objective parameters such as to a lesser extent. The treatment or amelioration of symptoms can be based on subjective or objective parameters, including the results of a medical examination. Thus, the term “treat” refers to the present invention for preventing or delaying, alleviating or preventing or inhibiting the progression of symptoms or conditions associated with a disease, condition or disorder as described herein. Administration of a compound or agent. The term “therapeutic effect” refers to the reduction, elimination or prevention of a disease, disease symptoms, or side effects of a disease in a subject. “Treat” or “treatment” using the methods of the invention is at increased risk of a disease or disorder associated with a disease, condition or disorder as described herein, but has not yet experienced or shown symptoms Preventing the onset of symptoms in the subject, suppressing the symptoms of the disease or disorder (delaying or preventing its progression), providing relief from symptoms or side effects of the disease (including symptomatic treatment), and (regression) Alleviating the symptoms of the disease. Treatment is prophylactic suppression (to prevent or delay the onset of the disease, or prevent their clinical or subclinical manifestations), or therapeutic suppression, or relief of symptoms after the onset of the disease or condition. Can be.

  `` Combination administration '' of a known drug (or other compound) and the composition of the present invention means that when both the known drug (or other compound) are considered to have a therapeutic or diagnostic effect, It means administering a drug (or other compound) as well as the composition. Such co-administration can include co-administration (ie, at the same time), pre-administration, or subsequent administration of the drug (or other compound) relative to the administration of the composition of the invention. One of ordinary skill in the art can determine the appropriate time, sequence, and dosage for administration of a particular drug (or other compound) and the composition of the present invention without difficulty.

  The terms “protection” and “prevention” are used interchangeably herein, meaning that infection by influenza is prevented.

  “Prophylactic vaccine” means that the vaccine prevents influenza infection upon future exposure.

  The term “immunogenic composition” or “composition” means that the composition is capable of inducing an immune response and is therefore antigenic. “Immune response” means any reaction by the immune system. These reactions include altering the activity of an organism's immune system in response to an antigen, and can include, for example, antibody production, induction of cell-mediated immunity, complement activation, or generation of immune tolerance.

  The term “disease” as used herein has a generally known and understood meaning in the art, and includes any abnormal condition in the functioning or satisfactory state of the host individual. Diagnosis of a specific disease by a medical professional can be made by direct examination and / or consideration of the results of one or more diagnostic tests.

  The terms “live vaccine composition”, “live vaccine”, “bacterial live vaccine”, and similar terms refer to a composition comprising a strain of live Bordetella bacteria that provides at least partial protective immunity against influenza.

  The terms "oral", "enteral", "enteral", "oral", "non-parenteral", "non-parenteral" and the like refer to a compound to an individual by a route or method along the digestive tract. Or refers to administration of the composition. Examples of “oral” routes of administration of the composition include swallowing the liquid or solid form of the vaccine composition by mouth, administration of the vaccine composition via the nasal jejunum or gastrostomy tube, intraduodenal administration of the vaccine composition And, for example, but not limited to rectal administration with suppositories that release a live bacterial vaccine strain described herein.

  The term `` topical administration '' refers to the application of a drug to the outer surface of the skin or mucosa (including the nasal, lung and mouth surface membranes) such that the agent crosses the outer surface of the skin or mucosa and enters the underlying tissue Point to. Topical administration can result in a limited distribution of the agent to the skin and surrounding tissue, or a systemic distribution of the agent if the agent is removed from the treatment area by the bloodstream. In a preferred form, the agent is delivered by transdermal delivery, for example using a transdermal patch. Transdermal delivery refers to the diffusion of an agent across the skin (the stratum corneum and epidermis), which functions as a barrier that the agent cannot penetrate. In contrast, the dermis is permeable to the absorption of many solutes and drugs, so topical administration is more easily performed through the skin where the epidermis has been abraded or otherwise exfoliated to expose the dermis. Is called. Absorption through untreated skin can be achieved by applying the active agent to an oily medium (e.g., emulsions, skins as described in Remington's Pharmaceutical Sciences, current edition, Gennaro et al., Eds.) Before application to the skin. It can be accelerated by combining with a softener, a penetration enhancer, etc. (a process known as rubbing).

  The term “nasal administration” means that the active ingredient is sprayed or otherwise introduced into the subject's nasal passage so that the active ingredient contacts the respiratory epithelium of the nasal cavity and is absorbed into the systemic circulation therefrom. Refers to any form of administration. Nasal administration can also include contacting the olfactory epithelium located in the upper part of the nasal cavity between the central nasal septum and the sidewalls of each of the main nasal passages. The area of the nasal cavity that directly surrounds the olfactory epithelium has no airflow. Thus, specialized methods typically must be used to achieve significant absorption across the olfactory epithelium.

  The term “aerosol” is used in the conventional sense to refer to very fine liquid or solid particles that are carried by a nebulizing gas under pressure to a treatment application site. The pharmaceutical aerosols of the present invention comprise a therapeutically active compound that can be dissolved, suspended or emulsified in a mixture of liquid carrier and propellant. Aerosols can be in the form of solutions, suspensions, emulsions, powders, or semi-solid preparations. The aerosols of the present invention are intended for administration as fine solid particles or liquid mists through the patient's airways. Various types of propellants may be used, including but not limited to hydrocarbon gas or other suitable gas. The aerosol of the present invention can also be delivered by a nebulizer that produces fine liquid particles of substantially uniform size within the gas. Preferably, the liquid containing the active compound is dispersed as droplets that can be carried into the patient's respiratory tract by the air stream exiting the nebulizer.

  The term “recover” refers to any therapeutically beneficial outcome in the treatment of disease states, eg, influenza-related disease states, including their prevention, reduction in severity or progression, remission or cure.

  In general, the phrase “show good tolerance” refers to the absence of adverse changes in health that occur as a result of treatment and affect treatment decisions.

  “Synergistic interaction” refers to an interaction in which the combined effect of two or more agents is greater than the algebraic sum of their individual effects.

  The term “in vitro” refers to a process performed on living cells that grow separately from living organisms, eg, living cells that grow in tissue culture.

  The term “in vivo” refers to a process performed in a living organism.

  The term “mammal” as used herein includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, dogs, cats, mice, cows, horses and pigs.

  The term “sufficient amount” means an amount sufficient to produce the desired effect, eg, an amount sufficient to modulate protein aggregation in the cell.

  The term “therapeutically effective amount” is an amount effective to ameliorate disease symptoms. A therapeutically effective amount can be a “prophylactically effective amount” because prophylaxis can be considered a treatment.

  As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or those Any other variation type is intended to cover non-exclusive inclusions. For example, a process, method, article, or device that includes a list of elements need not be limited to only those elements, but other elements that are not explicitly described or unique to such processes or methods. Can be included. Further, unless expressly specified otherwise, “or” refers to an inclusive “or” and not an exclusive “or”. For example, condition A or B is met by any one of the following: A applies (or exists) B does not apply (does not exist), A does not apply (or does not exist) B applies ( Present), and both A and B apply (or exist).

  It is to be understood that the present invention is not limited to a particular method, reagent, compound, composition, or biological system and can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” are not expressly specified otherwise. As long as it includes a reference to more than one. Thus, for example, reference to “a vaccine” includes a combination of two or more vaccines, and the like.

  As used herein, “about” when referring to a measurable value, such as amount, time, etc., is ± 20% or ± 10%, more preferably ± 5%, even more preferably ± from the specified value. It is intended to encompass variations of 1%, and more preferably ± 0.1% (variations suitable for performing the disclosed method).

Influenza virus types The present invention is generally used to treat or prevent influenza virus infection in mammals. There are three types of influenza viruses that can be targeted by the present invention: influenza A, B, and C. Influenza type A viruses are divided into subtypes based on two proteins on the surface of the virus. These proteins are termed hemagglutinin (H) and neuraminidase (N). Influenza A viruses are divided into subtypes based on these two proteins. 16 different hemagglutinin subtypes, H1, H2, H3, H5, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16, and 9 different neuraminidase subtypes, N1 , N2, N3, N4, N5, N6, N7, N8, or N9, all of which are found in wild bird influenza A viruses. Influenza A viruses include A (H1N1) and A (H3N2), both of which are examples of influenza viruses that can be targeted by the present invention for treatment or prevention in mammals. Diseases and symptoms typically caused by influenza virus infection may include the following: fever, cough, sneezing, pain, fatigue, headache, sulky eyes, nasal congestion, and abdominal pain. The present invention can be used to treat or prevent these diseases.

Composition
Bordetella strains The present invention provides mutant Bordetella strains that can be used as immunogenic compositions or vaccines to elicit an immune response in a mammal. In one aspect, the mutant Bordetella strain comprises a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene. The heterologous ampG gene product can significantly reduce the production of tracheal cytotoxins. In one aspect, the strain is BPZE1. The starting strain to be mutated can be any Bordetella strain, including B. pertussis, B. parapertussis, and B. septica. In one aspect, the starting strain used to obtain the mutant Bordetella strain is Bordetella pertussis. In another aspect, the strain is a triple mutant Bordetella strain. In another aspect, the Bordetella strain is identified by accession number CNCM I-3585. In another aspect, the Bordetella strain is identified by accession number V09 / 009169.

  The present invention is not limited only to the mutants described above. Other additional mutations can be made such as adenylate cyclase (AC) deficient mutants, lipopolysaccharide (LPS) deficient mutants, fibrillar hemagglutinin (FHA) and optional bvg regulatory system components.

  The construction of the mutant Bordetella strain of the present invention can begin by replacing the Bordetella ampG gene in the strain with a heterologous ampG gene. Any heterologous ampG gene known in the art can be used in the present invention. These examples can include all gram-negative bacteria that release very small amounts of peptidoglycan fragments per generation into the medium. Examples of gram-negative bacteria are Escherichia coli, Salmonella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, and Legionella. (Legionella) and the like, but not limited thereto. Typically, by replacing the Bordetella ampG gene with a heterologous ampG gene, the amount of tracheal cytotoxin (TCT) produced in the resulting strain exhibits less than 1% residual TCT activity. In another aspect, the amount of TCT toxin exhibited by the resulting strain is between about 0.6% and 1% residual TCT activity or between about 0.4% and 3% residual TCT activity or between about 0.3% and 5% Is the residual TCT activity between.

  PTX is a major virulence factor responsible for the systemic effects of Bordetella pertussis infection and is one of the major protective antigens. Because of its nature, the natural ptx gene is such that the enzymatically active component S1 encodes an enzymatically inactive toxin, but so that the immunogenic nature of pertussis toxin is not affected. It can be replaced with a mutant form. This can be achieved by replacing lysine (Lys) at position 9 of the sequence with arginine (Arg) (K9R). Furthermore, glutamic acid (Glu) at position 129 can be replaced with glycine (Gly) (E129G). In general, each of these amino acid positions is involved in substrate binding and catalysis. In other aspects, other mutations may also be made, such as the mutations described in US Pat. No. 6,713,072, incorporated herein by reference, and any known or other mutation that can reduce toxin activity. . In one aspect, allelic exchange can be used first to remove the ptx operon, and then the variant can be inserted.

  In another aspect of the invention, the dnt gene can be removed from the Bordetella strain using allelic exchange. Besides complete removal, enzyme activity can also be inhibited by point mutations. Since DNT is composed of a receptor-binding domain in the N-terminal region and a catalytic domain in the C-terminal part, a dnt point mutation that replaces Cys-1305 with Ala-1305 inhibits the enzyme activity of DNT ( Kashimoto T., Katahira J, Cornejo WR, Masuda M, Fukuoh A, Matsuzawa T, Ohnishi T, Horiguchi Y. (1999) Identification of functional domains of Bordetella dermonecrotizing toxin. Infect. Immun. 67: 3727-32.).

  In addition to allelic exchange that inserts a mutated ptx gene and a dnt gene that is inhibited or deleted, the open reading frame of the gene can be disrupted by insertion of a gene sequence or plasmid. This method is also contemplated in the present invention. Other methods for generating mutant strains are generally well known in the art.

  In one aspect of the present invention, the mutant strain is referred to as BPZE1 strain, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) in France, Paris under the Budapest Treaty on March 9, 2006, and numbered CNCM I-3585 is granted. Mutations introduced into BPZE1 generally result in attenuation, but also allow bacteria to colonize and continue to survive. Thus, in another aspect, the present invention provides BPZE1, and when administered to a mammal in need of BPZE1, BPZE1 can induce mucosal and systemic immunity. In another aspect of the invention, a BPZE1 recombinant strain expressing 3 copies of the M2e peptide was created. This strain was deposited with the National Measurement Institute (formerly AGAL) in Port Melbourne, Victoria, Australia 3207 under the Budapest Treaty on April 27, 2009 and has been given the accession number V09 / 009169. M2e is the extracellular part of the M2 protein from influenza virus. It is highly conserved among all influenza A viruses and has been shown to induce antibody-mediated protection against influenza A virus. Recombinant M2e-producing BPZE1 strain induces a substantial anti-M2e antibody response (locally and systemically) (for example, by nasal administration of live bacteria), and BPZE1 bacteria alone against H1N1 and H3N2 attacks Enables significant protection comparable to

  The mutant Bordetella strains of the present invention can be used in immunogenic compositions for the treatment or prevention of influenza virus infection. Such immunogenic compositions are useful for enhancing either an immune response, an antibody response in a mammal and / or a T cell response. For example, the T cell response may be such that the mammal is protected against influenza infection or its consequences / diseases / symptoms.

  The mutant Bordetella strain of the present invention can be used as a live strain in a vaccine or immunogenic composition. In one aspect, live strains can be used for nasal administration, while strains that have been killed chemically or by heating can be used for systemic or mucosal administration. In other aspects, the strain is attenuated.

  In another aspect of the invention, the strain does not contain any heterologous gene other than the heterologous ampG gene described above. In yet other aspects, the strain does not include a heterologous expression platform (see, eg, WO2007104451). Typically, a heterologous expression platform carries a heterologous antigen. In one aspect, a heterologous expression platform can be used to deliver a heterologous antigen to the mammalian respiratory mucosa.

Adjuvants The compositions of the present invention can be administered with other immunomodulatory agents including adjuvants. As used herein, the term “adjuvant” refers to a compound or mixture that enhances the immune response. Specifically, the composition can include an adjuvant. Adjuvants for use with the present invention can include, but are not limited to, one or more of the items described below.

Inorganic-containing adjuvant compositions Inorganic-containing compositions suitable for use as adjuvants in the present invention include inorganic salts such as aluminum and calcium salts. The present invention relates to hydroxides (eg, oxyhydroxides), phosphates (eg, hydroxyphosphoric acid, orthophosphoric acid), and sulfates (eg, Vaccine Design... (1995) eds. Powell & Newman ISBN: 030644867X. Plenum. See chapters 8 and 9) or a mixture of different inorganic compounds (eg phosphate and hydroxide, optionally with excess phosphate) The compound is in any suitable form (eg, gel, crystal, amorphous, etc.) and is preferably adsorbed onto the salt. Inorganic-containing compositions can also be formulated as metal salt particles (WO / 0023105).

Aluminum salts can be included in the compositions of the invention such that the dose of Al ³⁺ is between 0.2 mg and 1.0 mg per dose.

Oil Emulsion Adjuvant An oil emulsion composition suitable for use as an adjuvant in the present invention is MF59 (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into micron particles using a microfluidizer. ) And other squalene-water emulsions. See for example WO90 / 14837. See also Podda, "The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine", Vaccine 19: 2673-2680, 2001.

  In other related aspects, the adjuvant for use in the composition is a submicron oil-in-water emulsion. Examples of submicron oil-in-water emulsions for use herein include 4-5% w / v squalene, 0.25-1.0% w / v Tween 80 (polyoxyethylene sorbitan monooleic acid) and / or 0.25- 1.0% Span85 (sorbitan trioleate) and optionally N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxy Squalene / water emulsions optionally containing various amounts of MTP-PE, such as submicron oil-in-water emulsions containing phosphoryloxy) -ethylamine (MTP-PE), such as submicron oil-in-water emulsions known as `` MF59 '' Including emulsions (International Publication No. WO90 / 14837; US Pat. Nos. 6,299,884 and 6,451,325, which are incorporated herein by reference in their entirety; and Ott et al., “MF59--Design and Evaluation of a Safe and Potent” Adjuvant for Human Vaccines "in Vaccine Design: The Su bunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 can include 4-5% w / v squalene (eg, 4.3%), 0.25-0.5% w / v Tween 80 and 0.5% w / v Span 85, optionally with varying amounts of MTP-PE And is formulated into submicron particles using a microfluidizer such as a Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in an amount of about 0-500 μg / dose, or 0-250 μg / dose, or 0-100 μg / dose.

  Submicron oil-in-water emulsions, methods for making them, and immunostimulants such as muramyl peptides for use in compositions are detailed in International Publication Nos. WO90 / 14837 and US Pat. Nos. 6,299,884 and 6,451,325. Which are incorporated herein by reference in their entirety.

  Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants in the present invention.

Saponin adjuvant formulations Saponin formulations may also be used as adjuvants in the present invention. Saponins are a heterogeneous group of sterol glycosides and are triterpenoid glycosides that are also found in the bark, leaves, stems, roots and flowers of a wide range of plant species. Saponins from the bark of Quillaia saponaria Molina tree have been extensively studied as adjuvants. Saponins can also be obtained commercially from Smilax ornata (Salsaparilla), Gypsophilla paniculata (Bridal Veil), and Saponaria officianalis (Gypsophila). Saponin adjuvant formulations can include purified formulations such as QS21, as well as lipid formulations such as immune stimulating complexes (ISCOM; see below).

  Saponin compositions have been purified using high performance thin layer chromatography (HPLC) and reverse phase high performance liquid chromatography (RP-HPLC). Specific purified fractions have been identified using these techniques, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. The method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations can also contain sterols such as cholesterol (see WO96 / 33739).

  The combination of saponin and cholesterol can be used to form unique particles called ISCOMs. ISCOMs typically also include phospholipids such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOM. For example, ISCOM can include one or more Quil A, QHA and QHC. ISCOMs are further described in EP0109942, WO96 / 11711, and WO96 / 33739. Optionally, ISCOM can lack additional surfactant. See WO00 / 07621.

  A description of the development of saponin-based adjuvants can be found in Barr, et al., “ISCOMs and other saponin based adjuvants”, Advanced Drug Delivery Reviews 32: 247-27, 1998. See also Sjolander, et al., "Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines", Advanced Drug Delivery Reviews 32: 321-338, 1998.

Virosome and virus-like particles (VLP)
Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the present invention. These structures generally comprise one or more virus-derived proteins, optionally combined with or formulated with phospholipids. They are generally non-pathogenic, non-replicating and generally do not contain any natural viral genome. Viral proteins can be produced recombinantly or isolated from whole virus. These viral proteins suitable for use in virosomes or VLPs include influenza virus (such as HA or NA), hepatitis B virus (such as core protein or capsid protein), hepatitis E, measles virus, Sindbis virus, rotavirus From, foot-and-mouth disease virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA phage, QB phage (such as coat protein), GA phage, fr phage, AP205 phage, and Ty (such as retrotransposon Ty protein pl) Containing proteins.

Bacterial Derivatives or Microbial Derivatives Adjuvants suitable for use in the present invention include bacterial derivatives or microbial derivatives as follows.

(1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS) Such derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3 dMPL). 3 dMPL is a mixture of 3-O-deacylated monophosphoryl lipid A and 4, 5 or 6 acylated chains. An example of a “small particle” form of 3-O-deacylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such “small particles” of 3 dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics such as aminoalkyl glucosaminide phosphate derivatives such as RC-529. See Johnson et al., Bioorg Med Chem Lett 9: 2273-2278, 1999.

(2) Lipid A derivatives Lipid A derivatives can include derivatives of lipid A derived from E. coli such as OM-174. OM-174 is, for example, Meraldi et al., "OM-174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei ", Vaccine 21: 2485-2491, 2003; and Pajak, et al.," The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo ", Vaccine 21: 836-842, 2003. Yes.

(3) Immunostimulatory oligonucleotide An immunostimulatory oligonucleotide suitable for use as an adjuvant in the present invention has a CpG motif (a sequence containing unmethylated cytosine followed by guanosine and linked by a phosphate bond). A nucleotide sequence can be included. Bacterial double-stranded RNA or oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory.

  CpG can include nucleotide modifications / analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, guanosine can be replaced with an analog such as 2'-deoxy-7-deazaguanosine. Examples of analog substitutions are Kandimalla, et al., "Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles", Nucleic Acids Research 31: 2393-2400, 2003; WO02 / 26757 and See WO99 / 62923. The adjuvant effect of CpG oligonucleotides is described by Krieg, "CpG motifs: the active ingredient in bacterial extracts?", Nature Medicine (2003) 9 (7): 831-835; McCluskie, et al., "Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA ", FEMS Immunology and Medical Microbiology (2002) 32: 179-185; W098 / 40100; US Pat.No. 6,207,646; US Pat.No. 6,239,116 and US Pat. Further discussion at 6,429,199.

  CpG sequences can be directed to Toll-like receptors (TLR9) such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs", Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpG sequence can be specific to induce a Th1 immune response, such as CpG-A ODN, or can be more specific to induce a B cell response, such as CpG-B ODN. CpG-A ODN and CpG-B ODN are described in Blackwell, et al., "CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha", J. Immunol 170: 4061-4068, 2003; Krieg, “From A to Z on CpG”, TRENDS in Immunology 23: 64-65, 2002, and WO01 / 95935.

  In some aspects, the CpG oligonucleotide can be configured such that the 5 ′ end is available for receptor recognition. Optionally, two CpG oligonucleotide sequences can be joined at their 3 ′ ends to form an “immunomer”. For example, Kandimalla, et al., "Secondary structures in CpG oligonucleotides affect immunostimulatory activity", BBRC 306: 948-95, 2003; Kandimalla, et al., "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic See GpG DNAs ", Biochemical Society Transactions 31: 664-658, 2003; Bhagat et al.," CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents "BBRC 300: 853-861, 2003, and WO03 / 035836.

(4) ADP-ribosylating toxin and its detoxified derivatives Bacterial ADP-ribolysylated toxin and its detoxified derivatives can be used as adjuvants in the present invention. For example, the toxin can be derived from E. coli (ie, E. coli heat labile enterotoxin (LT)), cholera (CT), or pertussis (PTX). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95 / 17211 and parenteral adjuvants in WO98 / 42375. In some aspects, the adjuvant can be a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP ribosylating toxins and their detoxified derivatives, specifically LT-K63 and LT-R72 as adjuvants, is specifically described below, each of which is specifically incorporated herein by reference in their entirety. Can be found in the literature: Beignon, et al., "The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4 + T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin", Infection and Immunity 70: 3012-3019, 2002; Pizza, et al., "Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants", Vaccine 19: 2534-2541, 2001; Pizza, et al., "LTK63 and LTR72, two mucosal adjuvants ready for clinical trials "Int. J. Med. Microbiol 290: 455-461, 2003; Scharton-Kersten et al.," Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants ", Infection and Immunity 68 : 5306-5313, 2000; Ryan et al., "Mutants o f Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells "Infection and Immunity 67: 6270-6280, 2003; Partidos et al., "Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides", Immunol. Lett. 67: 09-216, 1999; Peppoloni et al., "Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines", Vaccines 2: 285-293, 2003; and Pine et al., (2002) "Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63) "J. Control Release 85: 263-270, 2002. The reference numbers for amino acid substitutions are preferably the A subunit and B subunit of the ADP-ribosylating toxin described in Domerighini et al., Mol Microbiol 15: 1165-1167, 1995, which is specifically incorporated herein by reference in its entirety. Based on unit sequence.

Bioadhesives and mucoadhesives Bioadhesives and mucoadhesives can also be used as adjuvants in the present invention. Suitable bioadhesives include ester-type hyaluronic acid microspheres (Singh et al., J. Cont. Rele. 70: 267-276, 2001) or poly (acrylic acid), polyvinyl alcohol, polyvinyl pyrrolidone, Mucoadhesive agents such as polysaccharides and cross-linked derivatives of carboxymethyl cellulose can be included. Chitosan and its derivatives can also be used as adjuvants in the present invention. See for example WO99 / 27960.

Adjuvant microparticle microparticles can also be used as adjuvants in the present invention. Formed from materials that are biodegradable and / or non-toxic (eg, poly (α-hydroxy acids), polyhydroxybutyric acid, polyorthoesters, polyanhydrides, and polycaprolactones) and poly (lactide-co-glycolide) Microparticles (i.e., particles with a diameter of about 100 nm to about 150 μm, or a diameter of 200 nm to about 30 μm, or a diameter of about 500 nm to about 10 μm) are envisaged, optionally (e.g. by SDS) It is treated to have a surface with a charge or a surface with a positive charge (for example by a cationic surfactant such as CTAB).

Examples of liposome formulations suitable for use as adjuvant liposome adjuvants are described in US Pat. No. 6,090,406, US Pat. No. 5,916,588, and EP 0 626 169.

I. Polyoxyethylene ether formulations and polyoxyethylene ester formulations Adjuvants suitable for use in the present invention also include polyoxyethylene ethers and polyoxyethylene esters. WO99 / 52549. Such a formulation comprises a polyoxyethylene sorbitan ester surfactant in combination with octoxynol (WO01 / 21207) and a polyoxyethylene alkyl ether or ester surfactant at least one additional agent such as octoxynol. It can be further included in combination with a nonionic surfactant (WO01 / 21152).

  In some aspects, the polyoxyethylene ether can include: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8- Steolyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, or polyoxyethylene-23-lauryl ether.

Polyphosphazene (PCPP)
PCPP formulations for use as adjuvants are described, for example, in Andrianov et al., "Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions", Biomaterials 19: 109-115, 1998, and Payne et al., "Protein Release from Polyphosphazene Matrices" , Adv. Drug. Delivery Review 31: 185-196, 1998.

Examples of muramyl peptides suitable for use as adjuvants in the muramyl peptides present invention, N- acetyl - muramyl -L- threonyl -D- isoglutamine (thr-MDP), N- acetyl - normuramyl-1-alanyl - d-isoglutamine (nor-MDP), and N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy ) -Ethylamine MTP-PE).

Examples of imidazoquinolone compounds suitable for use as adjuvants in imidazoquinolone compounds present invention can include Imiquimod and its homologs, Stanley, "Imiquimod and the imidazoquinolones : mechanism of action and therapeutic potential" Clin Exp Dermatol 27 : 571-577, 2002 and Jones, "Resiquimod 3M", Curr Opin Investig Drugs 4: 214-218, 2003.

Human immunomodulators Human immunomodulators suitable for use as adjuvants in the present invention include interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, and IL -12), cytokines such as interferons (eg, interferon gamma), macrophage colony stimulating factor, and tumor necrosis factor.

Combinations of adjuvants The present invention can also include combinations of one or more aspects of the adjuvants identified above. For example, the adjuvant composition can include:
(1) Saponin and oil-in-water emulsion (WO99 / 11241);
(2) saponins (eg QS21) + non-toxic LPS derivatives (eg 3dMPL) (see WO94 / 00153);
(3) saponins (eg QS21) + non-toxic LPS derivatives (eg 3dMPL) + cholesterol;
(4) Saponin (eg QS21) + 3dMPL + IL-12 (optionally + sterol) (WO98 / 57659);
(5) For example, a combination of QS21 and / or an oil-in-water emulsion and 3dMPL (see European patent applications 0835318, 0735898 and 0761231);
(6) Contains 10% squalane, 0.4% Tween 80, 5% pluronic block polymer L121 and thr-MDP, microfluidized or vortexed into submicron emulsions to yield larger grain size emulsions SAF;
(7) one or more of bacterial cell wall components derived from the group consisting of 2% squalene, 0.2% Tween 80, and monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS); Ribi adjuvant system (RAS) (Ribi Immunochem), preferably comprising MPL + CWS (Detox); and (8) one or more inorganic salts (such as aluminum salts) + non-toxic derivatives of LPS (such as 3dPML).

  Aluminum salts and MF59 are examples of adjuvants for use with injectable influenza vaccines. Bacterial toxins and bioadhesives are examples of adjuvants for use with mucosal delivery vaccines such as nasal vaccines. All of the above mentioned adjuvants and other adjuvants generally known to those skilled in the art can be formulated for intranasal administration using techniques well known in the art.

Formulations and methods for the treatment or prevention of diseases associated with carrier influenza virus infection (described in detail below) are also encompassed by the present invention. Said method of the invention comprises the step of administering a therapeutically effective amount of the composition of the invention. The composition of the present invention may be formulated into a pharmaceutical composition. These compositions can include, in addition to one or more strains, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should typically be non-toxic and typically should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, eg, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.

  Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Saline, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.

  For intravenous, dermal or subcutaneous injection, or at the site of disease, the active ingredient is a pyrogen-free and parenterally acceptable aqueous solution with suitable pH, isotonicity and stability It is a form. Those skilled in the art are well able to prepare suitable solutions using isotonic media such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

  Administration is preferably a “therapeutically effective amount” or “prophylactically effective amount” (although in some cases prophylaxis can be considered a treatment), this is sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. The prescription of treatment, eg determination of dosage etc., is within the responsibility of general practitioners and other physicians and is typically the disorder to be treated, the individual patient condition, the site of delivery, the method of administration and Consider other factors known to the practitioner. Examples of the techniques and protocols described above can be found in the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, PA (“Remington's”).

  Typically, the composition may be administered either alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated.

Method
Route of administration The compositions of the invention are generally administered directly to a mammal. Direct delivery can be by parenteral injection (e.g., subcutaneously, intraperitoneally, intradermally, intravenously, intramuscularly, or into the interstitial space of tissues) or by mucosal administration, e.g. rectal, oral ( (E.g. tablets, sprays), vaginal administration, topical administration, transdermal (see e.g. WO99 / 27961) or transcutaneous (e.g. see WO02 / 074244 and WO02 / 064162), inhalation, intranasal administration (e.g. For example, see WO03 / 028760), ocular administration, otic administration, pulmonary administration or other mucosal administration. The composition can also be administered topically by direct introduction to the surface of the skin. Topical administration can be accomplished without utilizing any device or by contacting the composition utilizing a bandage or bandage-like device with bare skin (see, eg, US Pat. No. 6,348,450).

  In some aspects, the mode of administration is parenteral immunity, mucosal immunity, or a combination of mucosal and parenteral immunity. In other aspects, the mode of administration is parenteral immunization, mucosal immunization, or a combination of mucosal and parenteral immunization with a total of 1-2 vaccinations separated by 1-3 weeks. In related aspects, the route of administration includes, but is not limited to, intranasal delivery.

Administration Methods and Dosages The present invention can include administration of a mutant Bordetella strain to a mammal to elicit an immune response (eg, a TH1 immune response) that can affect influenza viruses, such as H3N2. Examples of mutant Bordetella strains of the present invention are described above. Typically, administration of mutant Bordetella strains is used to treat or prevent influenza virus infection in mammals, eg, humans, through protective immunity against influenza virus. In some aspects, mutant Bordetella strain administration is used to prevent influenza infection by administration prior to influenza virus infection. Typically, mutant Bordetella strains are less than about 1 week, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks of influenza virus infection. Administered to mammals week, 12 weeks or more.

  In one aspect, a method for treating or preventing an infection with influenza virus comprises a single administration of a composition of the invention, eg, BPZE1, to a subject in need thereof. In a related aspect, the administering step is performed on the mucosa, eg, intranasally.

  In other aspects, the compositions of the invention are administered more than once, eg, twice. The number of administrations will vary as needed, for example, the number of administrations to a mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sell. In one aspect, a method for treating or preventing infection with influenza virus comprises administering to a subject in need thereof a first immunogenic composition of the invention (e.g. comprising BPZE1) followed by a second. Administering an immunogenic composition of (eg comprising BPZE1). Typically, the time range between each administration of the composition is about 1-6 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, It can be 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 weeks or longer. In a related aspect, the time range between each administration is about 3 weeks. In other aspects, a prime boost method can be used, wherein the composition of the present invention can be delivered in a “prime immunization” step and subsequently the composition of the present invention can be delivered in a “boost” step.

The composition can typically be used to elicit systemic and / or mucosal immunity, for example to elicit enhanced systemic and / or mucosal immunity. For example, the immune response can be characterized by induction of serum IgG and / or intestinal IgA immune response. Typically, the level of protection against influenza infection exceeds 50%, e.g. 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or more. In one aspect, the level of protection can be 100%. In other aspects, the level of protection is less than 50%, such as 20%. In other aspects, the number of bacteria in each dose is adjusted to achieve an effective immune response in the mammal. The number of bacteria or cfu in each dose is about 1, 10, 100, 1000, 10000, 100,000, 1000000, 5 × 10 ⁶ , or more, or any dose between each of these doses sell.

  In other aspects, the present invention can also include co-administration of the composition with one or more other agents. Typically, the various compositions / agents can be delivered in any order. Thus, in aspects involving delivery of multiple different compositions or agents, mutant Bordetella strains are agents that can affect influenza infection, such as drugs, siRNAs, miRNAs, immunogenic peptides, or small molecules. Not all need to be delivered before. Other examples of agents include neuraminidase inhibitors and M2 inhibitors (adamantanes). For example, priming can include delivery of one or more agents, and boosting can include delivery of one or more mutant Bordetella strains. In other aspects, multiple administrations of the mutant Bordetella strain can involve multiple administrations of the agent. Administration can be performed in any order. Thus, one or more mutant Bordetella strains and one or more agents described herein can be used in any order and in any route known in the art to elicit, for example, an immune response. Can be administered simultaneously.

  In the present invention, dosage treatment can be based on a single dose schedule or a multiple dose schedule. For example, multiple doses can be used in a primary immunization schedule and / or a booster schedule. In a multiple dose schedule, various doses can be given by the same or different routes, such as parenteral priming and mucosal boosting, mucosal priming and parenteral boosting. In other aspects, the dosing regimen can enhance the titer of the antibody response that results in an antibody having neutralizing properties. In vitro neutralization assays can be used to test neutralizing antibodies (eg Asanaka et al., J Virology 102: 10327, 2005; Wobus et al., PLOS Biology 2; e432; and Duubekti et al., J Medical Virology 66: 400).

One method of assessing the effectiveness of a test therapeutic treatment to determine the effectiveness or presence of an immune response includes monitoring infection after administration of the composition of the invention. One method of assessing the effectiveness of a prophylactic treatment involves monitoring an immune response to an antigen in the composition after administration of the composition of the invention. Another method of assessing the immunogenicity of the compositions of the invention is to isolate the protein or protein mixture and screen the patient's serum or mucosal secretions by immunoblotting. A positive reaction between the protein and patient serum indicates that the patient has already initiated an immune response to the composition.

  Another method of confirming the effectiveness of a therapeutic treatment involves monitoring the infection after administration of the composition of the invention. One way of confirming the effectiveness of prophylactic treatment is to administer a systemic immune response (such as monitoring the level of IgG1 and IgG2a production) to the antigen in the composition after administration of the composition of the present invention and mucosal immune response ( Monitoring both), such as producing levels of IgA production. Typically, serum specific antibody responses are determined after immunization but before challenge, while mucosal specific antibody responses are determined after immunization and after challenge. The immunogenic compositions of the invention can be evaluated in in vitro and in vivo animal models prior to administration to a host, eg, a human.

  The effectiveness of the compositions of the invention can also be determined in vivo by attacking an animal model of infection, such as a mouse, with the composition. The composition may or may not be derived from the same strain as the attacking strain. In vivo efficacy models can include, but are not limited to: (i) mouse infection models using human strains; (ii) mouse models using mouse-matched strains such as strains that are particularly pathogenic in mice. A mouse disease model that is: and (iii) a primate model using human isolates.

  The immune response induced by the present invention can be one or both of a TH1 immune response and a TH2 response. The immune response can be an improved immune response, an enhanced immune response, or an altered immune response. The immune response can be one or both of a systemic immune response and a mucosal immune response. For example, the immune response can be an enhanced systemic response and / or a mucosal response. Enhanced systemic and / or mucosal immunity is reflected in an enhanced TH1 immune response and / or TH2 immune response. For example, an enhanced immune response can include increased production of IgG1 and / or IgG2a and / or IgA. In another aspect, the mucosal immune response can be a TH2 immune response. For example, the mucosal immune response can include increased production of IgA.

  Typically, activated TH2 cells enhance antibody production and are therefore useful in responding to extracellular infections. Activated TH2 cells are typically capable of secreting one or more of IL-4, IL-5, IL-6 and IL-10. A TH2 immune response can also result in the production of IgG1, IgE, IgA, and / or immune memory B cells for future protection. In general, a TH2 immune response is an increase in one or more cytokines (such as IL-4, IL-5, IL-6 and IL-10) associated with a TH2 immune response, or IgG1, IgE, IgA and immune memory B One or more of the increased production of cells can be included. For example, an enhanced TH2 immune response can include an increase in IgG1 production.

  TH1 immune response is an increase in CTL, one or more of cytokines associated with TH1 immune response (such as IL-2, IFN-γ, and TNF-α), increased activated macrophages, increased NK activity, Alternatively, one or more of increased production of IgG2a can be included. For example, an enhanced TH1 immune response can include an increase in IgG2a production.

  Compositions of the invention, in particular immunogenic compositions comprising one or more strains of the invention, can optionally be Th1 responsive and / or Th2 either alone or in combination with other agents. It can be used with immunomodulators that can elicit a response.

  The compositions of the present invention can elicit both a cell-mediated immune response as well as a humoral immune response and effectively respond to influenza infection. This immune response preferably induces long-lasting (eg, neutralizing) antibodies and cell-mediated immunity that can respond rapidly upon exposure to one or more infectious antigens in the future.

Subjects and Mammals The compositions of the present invention are typically for preventing or treating influenza virus strains in mammalian subjects such as humans. In some aspects, the subject may be an elderly person (eg,> 65 years old), a child (eg, <5 years old), an inpatient, a healthcare worker, an army or soldier, a food handler, a pregnant woman, a chronically ill patient, and travel abroad. Can include people going out to. The compositions are generally suitable for these groups as well as other populations deemed necessary by the general population or physician.

Kits The present invention also provides kits comprising one or more containers of the composition of the present invention. The composition can be in liquid form or can be lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials including glass or plastic. The container can include a sterile access port (eg, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle).

  The kit further includes a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, or glucose solution. It can further include other materials useful to the end user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. . The kit can further include a third component that includes an adjuvant.

  The kit can also include a package insert containing instructions for a method of inducing immunity, a method of preventing an infection, or a method for treating an infection. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.

  The present invention also provides a delivery device pre-filled with the composition of the present invention.

  Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic and in full compliance with all US Food and Drug Administration Standards for Manufacturing and Quality Control (GMP) regulations Is done.

  From the foregoing description, various modifications and variations of the composition and method will occur to those skilled in the art. All such modifications that are within the scope of the appended claims are intended to be included within the scope of the claims. Each stated range includes all combinations and subcombinations of ranges, and the specific numbers contained therein.

  Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, those skilled in the art will recognize that certain variations and modifications will be understood by the present disclosure and shown by way of example and not limitation It will be apparent that it may be practiced without undue experimentation within the scope of the claims.

Exemplary Aspects The following are examples of specific aspects for carrying out the present invention. The examples are provided for illustrative purposes only and are in no way intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but of course some experimental error and deviation should be allowed for.

The practice of the present invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacy within the skill of the art, unless otherwise indicated. Such techniques are explained fully in the literature. For example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition , 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's; Carey and Sundberg Advanced Organic Chemistry 3 ^rd Ed. (Plenum Press) Volumes A and B, 1992). That.

Materials and Methods Bacterial strains and growth conditions The bacterial strains used in this study are skin necrotic (DNT) codes that produce inactive forms of pertussis toxin (PT) and background levels of tracheal toxin (TCT). Bordetella pertussis BPZE1, a streptomycin-resistant TohamaI derivative lacking the gene (22). BPZE1 bacteria were cultivated in Borde Jung (BG) agar (BG) supplemented with 1% glycerol, 10% sheep defibrinated blood and 100 μg / ml streptomycin (Sigma Chemical CO., St Louis, Mo.) for 72 hours at 37 ° C. Difco, Detroit, Mich.). As previously described (Menozzi FD, et al., "Identification and purification of transferring- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica", Infect, Immun 59: 3982-3988, 1991), 1 g / l Liquid culture was performed in Stainer-Scholte (SS) medium containing heptakis (2,6-di-o-methyl) β-cyclodextrin (Sigma). Heat deactivation was performed at 95 ° C. for 1 hour.

Intranasal infection
6-8 week old female Balb / c mice are maintained in individually ventilated cages under conditions free of specific pathogens and all experiments are conducted at the National University of Singapore Singapore animal study board). For BPZE1 treatment, as previously described (Mielcarek N, et al., "Intranasal priming with recombinant Bordetella pertussis for the induction of a systemic immune response against a heterologous antigen", Infect immune 65: 544-550, 1997 ), 20 μl sterile PBS (PBST) supplemented with 0.05% Tween80 (Sigma) containing approximately 5 × 10 ⁶ colony forming units (cfu) of live or dead bacteria of BPZE1 Administered intranasally once, twice or three times (as indicated). For influenza infection, mice given sedation were given approximately 2 × 10 ⁶ TCID ₅₀ mouse-adapted A / Aichi / 2/68 (H3N2) virus (passage number) in 20 μl sterile PBS supplemented with penicillin and streptomycin. 10) (Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis", Microbes Infect 11: 2-11, 2009) or 4 lethal doses ( LD) 50 H1N1 (A / PR / 8/34) influenza virus (ATCC # VR-95) was administered intranasally. Survival based on weight loss was determined using 10 mice per group, and mice were euthanized when the body weight decreased more than 20% of the original body weight.

Determination of the virus titer Mouse lungs are collected, homogenized using a mechanical homogenizer (Omni homogenizer), and a modified method reported by WHO (WHO, "WHO Manual on Animals Influenza Diagnosis and Surveillance" (World Health Organization, Geneva ), 2002) was tested for the presence of viable virus by a 50% tissue culture infectious dose (TCID ₅₀ ) assay. Briefly, 100 μl of lung homogenate serially diluted 10-fold was seeded in 90% confluent Maidin Derby canine kidney (MDCK) cells in 96-well plates. Plates were incubated for 3 days at 35 ° C. in a humidified incubator (5% CO ₂ ). TCID ₅₀ was determined by a 50% reduction in cytopathic effect (CPE) and led log TCID ₅₀ / lung. Five mice per group were evaluated individually at each time point.

Histopathology Three days after virus challenge, 4 mice per group were sacrificed and their lungs were collected. Lungs were removed and fixed in PBS containing 10% formalin. After fixation, the lungs were embedded in paraffin, cut into slices and stained with H & E.

Cell infiltration in bronchoalveolar lavage fluid (BALF) Inject 1 ml of sterile PBS into the lungs of a sacrificed animal and perform one lavage step to ensure that both lungs expand during processing Collected individual BALF. The BALF was then centrifuged at 400 g for 10 minutes and the supernatant was removed and stored at −80 ° C. for cytokine detection. The total number of BALF cells was determined using a hemocytometer. Cells were spotted on a slide glass using a cytospin apparatus (Thermo Shandon) and stained with a modified Wright staining method (Kimman TG, et al., "Development and antigen specificity of the lymphoproliferation response of pigs to pseudorabies virus: dichotomy between secondary B- and T-cell responses ", Immunology 86: 372-378, 1995). Different cell types were identified using standard morphological criteria. Results are expressed as a percentage of each cell relative to the total cell population. A total of 500 cells were examined per slide. Four mice per group were evaluated individually.

FACS analysis Mice were sacrificed and their lungs collected and lungs in 2 ml digestion buffer containing 0.5 mg / ml Liberase (Roche) in RPMI with 1% FCS and 2 U / ml DNaseI (Qiagen) Was digested at 37 ° C. for 15 minutes, and centrifuged at 600 g at room temperature for 20 minutes using Ficoll-PaqueTMPLUS (GE) to prepare a single cell suspension. Cells were collected and washed twice with sterile FACS buffer (2% FCS, PBS containing 5 mM EDTA). 10 ⁶ cells were stained with FITC-labeled anti-mouse CD3 antibody (eBioscience) and analyzed with a CyAn ™ ADP hemocytometer (Dako). Five mice per group were evaluated individually at each time point.

Analysis of cytokines and chemokines
Cytokine and chemokine production in the BALF supernatant was measured using the Procarta cytokine profiling kit according to the manufacturer's instructions (Panomics). After incubation with Ab-bound beads, detection Ab and streptavidin-PE complex, the sample was run on a Bio-Plex instrument (Bio-Rad). The levels of the following growth factors, cytokines, and inflammatory mediators were evaluated: GM-CSF, KC, IL-1β, IL-6, IFN-γ and TNF-α, IFN-α, MCP-1, RANTES, IL- Ten. In addition, TGF-β levels were measured using a human / mouse TGFβ1 ELISA kit (eBioscience) according to the manufacturer's instructions.

Passive transfer experiments High-titer anti-pertussis immune sera were raised in 10 adult Balb / c mice that were intranasally infected with 5 × 10 ⁶ cfu viable BPZE1 bacteria twice at 4 week intervals. Another group of 10 adult naïve Balb / c mice received 10 ^5.5 TCID ₅₀ of heat-inactivated human / Aichi / 2/68 (H3N2) virus (HI-H3N2) complete Freund's adjuvant intraperitoneally ( ip.) and injected 2 weeks later with an incomplete Freund's adjuvant containing the same amount of HI-H3N2 virus. Immune sera from each group of mice were collected 2 weeks after the booster, pooled, and anti-pertussis antibody titers and anti-influenza antibody titers were measured by ELISA. In addition, HI-H3N2 serum was tested for the presence of neutralizing antibodies by a neutralization assay. The immune serum was sterilized by filtration, heat inactivated at 56 ° C. for 30 minutes, and stored at −80 ° C. for future use. Serum from control untreated mice was also collected as a negative control.

  Recipient Balb / c mice 6-8 weeks old were injected ip with 200 μl of untreated immune serum, anti-BPZE1 immune serum, or anti-H3N2 immune serum one day prior to viral challenge with mouse-matched H3N2 virus. Weight loss was monitored and survival rate was determined. Ten mice per group were assayed.

T cell proliferation assay Lymphocyte proliferation as described elsewhere (Bao Z, et al., "Glycogen synthase kinase-3beta inhibition attenuates asthma in mice", Am J Respir Crit Care Med 176: 431-438, 2007), measured by tritiated ( ³ H) thymidine incorporation. Briefly, spleens from untreated and BPZE1-treated mice (6 mice per group) were collected under aseptic conditions and pooled. Single cell suspensions were prepared and centrifuged with Ficoll-PaqueTMPLUS (GE) at 600 g for 20 minutes at room temperature. Isolated splenocytes were placed in a 96-well round bottom plate (NUNC) in 100 μl medium (10% FCS, 5 × 10 ⁻⁵ M β-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES, 200 U / RPMI640 supplemented with ml penicillin and 200 μg / ml streptomycin) was seeded at a concentration of 2 × 10 ⁵ cells / well. 100 μl of medium containing 20 μg / ml BPZE1 whole cell lysate or heat inactivated 10 ⁵ TCID ₅₀ mouse-adapted H3N2 influenza virus (HI-H3N2) (test antigen) was added to splenocytes. 100 μl of medium containing 100 μl of uninfected egg amniotic fluid and 5 μg / ml concanavalin A (conA) were used as sham and survival controls, respectively. After 3 days of incubation at 37 ° C. in a 5% CO ₂ atmosphere, 20 μl RPMI complete medium containing 0.4 μCi [ ³ H] thymidine was applied to the culture. After 18 hours of incubation, cells were harvested, washed, and incorporated radioactivity was measured with a TopCount NXT ™ microplate scintillation luminescence counter (PerkinElmer). Results show stimulation index (SI) corresponding to the ratio between the mean [ ³ H] thymidine incorporation in the presence of test antigen and the mean [ ³ H] thymidine incorporation in the absence of test antigen Represented as: SI> 2 is considered positive. Each sample was assayed in quadruplicate.

IFN-α ELISPOT Assay The frequency of antigen-specific IFN-γ producing splenocytes was determined by ELISPOT assay using a BD mouse ELISPOT set (BD PharMingen) according to the manufacturer's instructions. Briefly, 96-well microplates prepared from single-cell suspensions of individual spleens from untreated and BPZE1-treated mice and pre-coated with 100 μl of sterile PBS containing 5 μg / ml anti-IFN-γ antibody (Millipore, Bedford, Mass.) Overnight at 4 ° C., washed 3 times and blocked with RPMI 1640 containing 10% FCS for 2 hours at room temperature. Cells were then combined with 20 μg / ml BPZE1 whole cell lysate, or heat inactivated 10 ⁵ TCID ₅₀ mouse-adapted H3N2 influenza virus (HI-H3N2), or 5 μg / ml conA with 5% CO _2. Incubate at 37 ° C. for 12-20 hours in an atmosphere. The plates were then washed and subsequently biotin-conjugated anti-mouse IFN-γ antibody was added for 2 hours at room temperature. After washing, streptavidin-HRP complex was added and incubated for 1 hour at room temperature. The wells were washed again and developed with 3-amino-9-ethyl-carbazole (AEC) substrate solution until spots were visible. After drying, the number of cells that formed spots was counted with a Bioreader® 4000 (Biosystem). Six animals per group were assayed individually.

Statistical analysis, unless otherwise specified, bars represent mean ± SD, mean two-way independent student with 5% significance level of ^* p ≤ 0.05, ^** p ≤ 0.01 and ^*** p ≤ 0.001 Comparison was made using an assay.

Example 1: A single nasal administration of live attenuated Bordetella pertussis prevents H3N2 influenza challenge.
Mouse-adapted H3N2 influenza virus was obtained through continuous passage from lung to lung of A / Aichi / 2/68 (H3N2) virus into adult Balb / c mice (Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis ", Microbes Infect 11: 2-11, 2009). Passage 10 (P10) strains were highly pathogenic and resulted in extrapulmonary spread with necrotic and inflammatory lesions in various organs of infected animals. 2 × 10 nasal administration of P10 virus suspension of ⁶ TCID ₅₀ has resulted in the death of the animals within 4 days (Narasaraju T, et al, " Adaptation of human influenza H3N2 virus in a mouse pneumonitis model:. Insights into viral virulence, tissue tropism and host pathogenesis ", Microbes Infect 11: 2-11, 2009).

  Adult Balb / c mice were inoculated nasally with live BPZE1 bacteria and challenged with a lethal dose of mouse-adapted H3N2 virus either 3 or 6 weeks later. Survival based on body weight change is not significantly protected in mice challenged 3 weeks after nasal BPZE1 treatment, while achieving 60% protection when mice are challenged 6 weeks after BPZE1 treatment (Fig. 1).

Example 2: BPZE1 live bacteria prevent lethal H3N2 attack, but BPZE1 dead bacteria do not.
Adult Balb / c mice were given a single nasal dose of BPZE1 live bacteria or BPZE1 dead bacteria and then challenged with a lethal dose of mouse-adapted H3N2 virus 6 weeks after BPZE1 treatment. The results showed that dead bacteria did not provide any significant protection against H3N2 (Figure 2), suggesting that bacterial colonization of the mouse lung is necessary to induce a protective mechanism.

Example 3: Effect of booster immunization.
Prior to lethal challenge with mouse-adapted H3N2 virus 4 weeks after the last BPZE1 administration, live BPZE1 bacteria were administered nasally to Balb / c mice twice at 4-week intervals. A 100% protection rate with minimal weight change was obtained for BPZE1-treated animals (Figure 3). Similar protection rates were achieved when the viral challenge was performed 2 weeks after the boost. These data indicated that the second nasal administration of BPZE1 live bacteria not only enhanced the protective effect but also reduced the time required to elicit the protective mechanism.

Example 4: BPZE1 bacteria provide protection against H1N1 virus attack.
We further explored the protective ability of BPZE1 bacteria against influenza A virus. Mice treated nasally with live BPZE1 bacteria were not protected against a lethal challenge with human A / PR / 8/34 (H1N1) influenza A virus 6 weeks later (data not shown) ). However, three consecutive doses of live BPZE1 bacteria provided 50% protection against H1N1 virus (FIG. 4). These observations indicated that BPZE1 bacteria may have the potential to protect against all influenza A viruses, although their effectiveness is variable.

Example 5: Viral load is not reduced in protected mice.
To further characterize cross-protection against influenza A virus, the viral load in the lungs of mice treated twice with BPZE1 bacteria and control mice was quantified. The viral load was confirmed 3 days after infection corresponding to the peak of viral titer in mice infected with mouse-adapted H3N2 virus (Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis ", Microbes Infect 11: 2-11, 2009). No significant difference in viral load was observed between the two groups of animals (Figure 5). This result suggested that the cross-protection mechanism elicited in BPZE1-treated animals does not directly target viral particles and / or infected cells.

Example 6: BPZE1 treatment protects mice from immune symptoms and lymphocyte depletion induced by influenza.
Lung immune symptoms were examined by histology of lung sections from infected BPZE1-treated animals and control mice. As expected and as previously described (Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis", Microbes Infect 11: 2-11, In 2009, infected control mice had severe inflammation with inflammatory cells, severe bronchial and interstitial pneumonia with bronchioles and alveoli filled with necrotic debris, and severe emphysema And moderate edema (FIG. 7A). In contrast, in the lungs of protected BPZE1-treated mice, only mild inflammation, minor airway and alveolar injury, and mild perivascular / peribronchial injury associated with mild edema were observed. .

Cell populations present in bronchoalveolar lavage fluid (BALF) recovered from protected and unprotected animals were also examined. The total number of cells present in BALF from both animal groups was comparable (11.8 × 10 ⁵ vs 16.1 × 10 ⁵ ), but BPZE1-treated mice had significantly more macrophages and fewer neutrophils (Figure 6B).

In addition, lymphocyte populations present in the lungs of protected and unprotected mice were analyzed. Mice infected with highly pathogenic H1N1 (1918) and H5N1 influenza viruses (Kash JC, et al., "Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus", Nature 443: 578-581, 2006 ; Uiprasertkul M, et al., "Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans", Emerg Infect Dis 13: 708-712, 2007; Lu X, et al., "A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans ", J Virol 73: 5903-5911, 1999), and mice infected with the mouse-compatible H3N2 virus strain used in this study (Narasaraju T, et al.," Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis ", Microbes Infect 11: 2-11, 2009). Here, Balb / c mice were treated twice with BPZE1 bacteria and challenged with mouse-adapted H3N2 virus after 4 weeks. Mouse lungs were collected 3 and 5 days after influenza challenge for FACS analysis of T cell populations. Three days after viral challenge, the percentage of CD3 ⁺ T lymphocytes in infected control and BPZE1-treated mice was comparable to that seen in pre-challenge animals (FIG. 6C). However, as previously reported (Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis", Microbes Infect 11: 2-11, 2009), significant CD3 ⁺ T cell depletion was observed in infected control animals 5 days after virus challenge (FIG. 6C). In contrast, in protected BPZE1 immunized animals, the T cell population was constant before and after challenge (FIG. 6C), suggesting that BPZE1 treatment prevented lymphocyte depletion induced by influenza.

Example 7: Production of pro-inflammatory cytokines and chemokines is suppressed in protected BPZE1-treated mice.
Major pro-inflammatory cytokines and chemokines were measured with BALF recovered from protected BPZE1-treated mice 1 and 3 days after lethal H3N2 virus challenge and compared to unprotected mice. All pro-inflammatory cytokines and chemokines measured with BALF in the protected animal group were significantly less than those measured in unprotected mice (FIG. 7). Day 1 and 3 for IL-1β, IL-6, IFN-γ, and TNF-α, the major pro-inflammatory cytokines that contribute to influenza-induced immune symptoms and correlate with disease severity Differences were observed in both (de Jong MD, et al., "Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia", Nat Med 12: 1203-1207, 2006; Beigel JH, et al., "Avian influenza A (H5N1) infection in humans :, N Engl J Med 353: 1374-1385, 2005; Peiris JS, et al.," Re-emergence of fatal human influenza A subtype H5N1 disease ", Lancet 363 : 617-619, 2004; Schmitz N, et al., "Interleukin-1 is responsible for acute lung immunopatholoy but increases survival of respiratory influenza virus infection", J Virol 79: 6441-6448, 2005) .IFN-α, MCP -1 and RANTES levels were significantly reduced 1 day after viral challenge, while IL-12 (p70), GM-CSF and KC production declined 3 days after challenge. , In protected fauna observed complete inhibition of IL-12 production was consistent with the decreased levels of IFN-gamma.

  Furthermore, no significant differences in the levels of anti-inflammatory cytokines IL-10 and TGF-β were detected between protected and unprotected animals, and type 1 regulatory T cells (Tr1) in a cross-protection mechanism Involvement was excluded (Figure 7).

Example 8: Bordetella pertussis specific adaptive immunity is not involved in cross-protection.
The presence of cross-reactive (and protective) antibodies and / or T cells in BPZE1-treated animals was examined. First, no matching epitope between B. pertussis and influenza A H3N2 and H1N1 viruses was identified by BLAST search (data not shown). Second, immune sera from BPZE1-treated mice did not react with H3N2 whole virus particles in an ELISA assay and did not neutralize virus in an in vitro neutralization assay (data not shown). Third, in in vivo passive transfer experiments, high titer anti-BPZE1 immune sera did not confer any protection against H3N2 lethal attack, but 100% of immune sera raised against heat-inactivated H3N2 virus Protection rate was given (FIG. 8A). Fourth, proliferation and IFN-γ ELISPOT assays showed that splenocytes from BPZE1-treated mice did not proliferate and produce IFN-γ, respectively, upon stimulation with H3N2 virus particles (FIGS. 8B and C). Overall, these data strongly support that Bordetella pertussis-specific immunity does not play any role in cross-protection against influenza A virus.

Discussion Severe respiratory disease and immune symptoms with high mortality are prominent features of highly pathogenic avian influenza virus infection in humans and other mammalian species. However, the underlying mechanisms that cause severe immune symptom effects have not yet been fully elucidated. Specifically, the relationship between viral load, immune symptoms, and disease outcome remains unclear. Several previous studies have reported reduced mortality and immune symptoms without significant reduction in viral load in animal models of influenza infection; for example, inflammatory cells in mice with disrupted MIP-1α gene It has been observed that viral invasion and lung damage are reduced but viral clearance is delayed (Cook DN, et al., "Requirement of MIP-1 alpha for an inflammatory response to viral infection", Science 269: 1583-1585, 1995 ). Similarly, mice lacking CCR2 (the main receptor for MCP-1) exhibited reduced mortality but a significant increase in viral load associated with reduced lung cell infiltration and tissue damage (Dawson TC, et al., “Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus”, Am J Pathol 156: 1951-1959, 2000). In contrast, IL-1R knockout mice showed increased mortality associated with delayed viral clearance but fewer severe symptoms (Schmitz N, et al., "Interleukin-1 is responsible for acute lung immunopatholoy but increases survival of respiratory influenza virus infection ", J Virol 79: 6441-6448, 2005).

  Here we report that pertussis-mediated cross-protection against influenza A virus does not result in reduced viral load in the lung. Instead, protected BPZE1-treated mice exhibited minimal pulmonary immune symptoms and reduced production of major pro-inflammatory cytokines and chemokines. Our findings are consistent with the general consensus that disease severity is strongly correlated with high cytokine / chemokine levels (La Gruta NL, et al, "A question of self-preservation: immunopathology in influenza virus infection ", Immunol Cell Biol 85: 85-92 2007). A cytokine storm, characterized by excessive levels of chemokines and cytokines in the serum and lungs by uncontrolled activation of the host immune system, is a laboratory animal infected with reconstituted 1918 H1N1 and H5N1 influenza viruses (Kash JC, et al. , "Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus", Nature 443: 578-581, 2006; Simon AK, et al., "Tumor necrosis factor- related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes ", Proc Natl Acad Sci USA 98: 5158-5163, 2001; Kobasa O, et al.," Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus ", Nature 445: 319-323, 2007; Tumpey TM, et al., "Characterization of the reconstructed 1918 Spanish influenza pandemic virus", Science 310: 77-80, 2005), and humans (de Jong MD, et al., "Fatal outcome of human influenza A ( H5N1) is associated with hi gh viral load and hypercytokinemia ", Nat Med 12: 1203-1207, 2006; Beigel JH, et al.," Avian influenza A (H5N1) infection in humans :, N Engl J Med 353: 1374-1385, 2005; Uiprasertkul M , et al., "Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans", Emerg Infect Dis 13: 708-712, 2007; Peiris JS, et al., "Re-emergence of fatal human influenza A subtype H5N1 disease ", Lancet 363: 617-619, 2004). Furthermore, histological and pathological indicators strongly suggest an important role played by excessive host responses in mediating at least some extreme pathologies associated with highly pathogenic influenza viruses. However, the role of each individual cytokine during influenza infection often has both positive and negative effects and is still unclear; their production mobilizes immune effector cells to the site of infection and / or Although they can be important for virus clearance through activation, their inflammatory properties can also lead to tissue damage (La Gruta NL, et al., "A question of self-preservation: immunopathology in influenza virus infection", Immunol Cell Biol 85: 85-92 2007).

Decreased proinflammatory cytokines and chemokine production in the respiratory organs of protected BPZE1-treated animals may have affected cell invasion and immune cell activation; neutrophil count in BALF of protected animals A significant decrease was actually observed, consistent with a decrease in the levels of KC and TNF-α (both cytokines are involved in neutrophil recruitment and activation in infected tissues) (La Gruta NL, et al., "A question of self-preservation: immunopathology in influenza virus infection", Immunol Cell Biol 85: 85-92 2007; Kips JC, et al., "Tumor necrosis factor causes bronchial hyperresponsiveness in rats", Am Rev Respir Dis 145: 332-336, 1992; Headley AS, et al., "Infections and the inflammatory response in acute respiratory distress syndrome", Chest 111: 1306-1321, 1997). In addition, suppression of IL-12 production measured in protected animals may have reduced activation of some immune cells such as natural killer (NK) cells and cytotoxic CD8 ⁺ T cells . Both cell types have been described as potentially harmful and involved in immunopathology related to the release of inflammatory mediators (La Gruta NL, et al., "A question of self-preservation: immunopathology in influenza virus infection ", Immunol Cell Biol 85: 85-92 2007). Consistently, IFN-γ production, a hallmark of NK and CD8 ⁺ T cell activation, was significantly reduced in BALF recovered from protected animals. In addition, reduced IFN-γ production can affect neutrophil responses to viruses, including oxidative bursts, induction of antigen presentation, and chemokine production (Ellis TN and Beaman BL, “Interferon- gamma activation of polymorphonuclear neutrophil function ", Immunology 112: 2-11, 2004; Farrar MA and Schreiber RD," The molecular cell biology of interferon-gamma and its receptor ", Annu Rev Immunol 11: 571, 1993).

  Interestingly, significantly higher numbers of macrophages are observed in BALF of protected BPZE1-treated mice despite low levels of MCP-1 and GM-CSF, which are involved in monocyte recruitment and differentiation into macrophages, respectively It was done. On the other hand, it should be noted that alveolar macrophages (AM) and non-tissue resident macrophages constitute the major macrophage population recovered by BALF (Jakubzick C, et al., "Modulation of dendritic cells trafficking to and from the airways ", J Immunol 176: 3578-3584, 2006). Therefore, it is believed that protected mice compared to infected control mice may exhibit a decrease in macrophage population in those tissues.

  AM regulates the function of T cells (Strickland DH, et al., "Regulation of T-cell function in lung tissue by pulmonary alveolar macrophages", Immunology 80: 266-272, 1993). By suppressing maturation (Holt PG, et al., "Down-regulation of the antigen presenting function (s) of pulmonary dendritic cells in vivo by resident alveolar macrophages", J Exp Med 111: 397-407, 1993; Bilyk N and Holt PG, "Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte / macrophage colony stimulating factor", J Exp Med 111: 1773-1777 ', 1993; Stumbles PA, et al., "Airway dendritic cells: co -ordinators of immunological homeostasis and immunity in the respiratory tract ", APMIS 111: 741-755, 2003) and by inhibiting migration to mesenteric lymph nodes (Jakubzick C, et al.," Modulation of dendritic cells trafficking to and from the airways ", J Immunol 176: 3578-3584, 2006), suppression of inflammatory reactions. It has been shown to exhibit the effect. Therefore, it can be hypothesized that a large number of alveolar macrophages induced by influenza challenge contributed to the suppression / control of inflammation in protected BPZE1-treated animals.

Furthermore, we found no significant change in the CD3 ⁺ T cell population in the protected BPZE1-treated animals, but a significant reduction in the ratio of CD3 ⁺ T cells in the infected control mice upon viral challenge. I found out. Lymphocyte depletion during highly pathogenic influenza infection has been reported previously (Kash JC, et al., "Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus", Nature 443: 578-581 , 2006; Uiprasertkul M, et al., "Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans", Emerg Infect Dis 13: 708-712, 2007; Narasaraju T, et al., "Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis ", Microbes Infect 11: 2-11, 2009; Lu X, et al.," A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans ", J Virol 73: 5903-5911, 1999), experimental evidence suggests apoptosis as a possible mechanism (Uiprasertkul M, et al.," Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans ", Emerg Infect Dis 13: 708-712, 2007; Lu X, et al.," A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans ", J Virol 73: 5903-5911, 1999). Lymphocyte apoptosis cannot be a direct cytolytic effect of the virus itself, as no difference in viral load is observed between protected and unprotected animals. Instead, our data are consistent with previous studies suggesting that cytokine regulation and overactivation of the host immune response may cause lymphocyte apoptosis in humans and mice infected with H5N1 (Uiprasertkul M, et al., "Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans", Emerg Infect Dis 13: 708-712, 2007; Maines TR, et al., "Pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response ", Immunol Rev 225: 68-84, 2008). Specifically, TNF-α and related TNF superfamily members, including TNF-related apoptosis-inducing ligand (TRAIL), are known to induce T cell apoptosis (Simon AK, et al., “Tumor necrosis factor -related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes ", Proc Natl Acad Sci USA 98: 5158-5163, 2001; Wang J, et al.," The critical role of LIGHT, a TNF family member, in T cell development ", J Immunol 167: 5099-5105, 2001). Consistently, low levels of TNF-α were measured in BALF of BPZE1 mice protected against influenza challenge, potentially leading to reduced T cell apoptosis.

  The protection mechanism responsible for cross defense has not yet been identified. However, our data demonstrate that pertussis-specific adaptive immunity (including cross-reactive antibodies and T cells) is not involved. The observation that BPZE1 live bacteria confer protection but not BPZE1 dead bacteria indicates that bacterial pulmonary colonization, ie, long-term exposure to the host immune system, is necessary to induce a protective mechanism. Furthermore, induction of the protective mechanism requires more than 3 weeks to occur, since mice treated once with live BPZE1 and challenged with influenza H3N2 virus after 3 weeks were not significantly protected. However, the second BPZE1 treatment can reduce the time required to induce a protection mechanism and enhance the protection rate, which is the first time some immune memory cells can respond faster. It was produced during immunization, suggesting that most immune memory cells were produced during the second encounter with BPZE1 bacteria. Finally, it has been observed that three consecutive nasal administrations of live BPZE1 bacteria are necessary to confer 50% protection against human A / PR / 8/34 (H1N1) influenza virus. The difference in protection rates achieved against H3N2 and H1N1 virus attacks suggests that the molecular disease mechanisms induced by both viruses are different. However, although protective efficacy can vary between subtypes, Bordetella pertussis is a promising universal vaccine against influenza A virus.

  Much of the activity of Bordetella pertussis virulence factors is devoted to immunomodulation to suppress, destroy and avoid host defense systems (Carbonetti NH, "Immunomodulation in the pathogenesis of Bordetella pertussis infection and disease", Curr Opin Pharmacol 7: 1-7, 2007). The immune response against Bordetella pertussis is initiated and controlled through Toll-like receptor (TLR) 4 signaling, an anti-inflammatory cytokine IL- by dendritic cells (DC) that can inhibit inflammatory responses and limit symptoms in the respiratory tract 10 Higgins SC, et al., "Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology", J Immunol 171: 3119- 3127, 2003). Filamentous hemagglutinin (FHA), a major attachment factor produced by Bordetella pertussis, stimulates IL-10 production, inhibits TLR-induced IL-12 production by macrophages and DCs, and secretes IL-10 (GcGuirk P, et al., "Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis ", J Exp Med 195: 221-231, 2002). Interestingly, systemic administration of FHA has recently been found to reduce enteritis in a T cell-mediated colitis model, which supports the anti-inflammatory role of FHA (Braat H, et al., "Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomoculatory molecule ", Gut 56: 351-357, 2007). However, in the present invention, there is no difference in the ratio of IL-10 and TGF between protected and unprotected mice, mediated by FHA in cross-protection against influenza A virus conferred by Bordetella pertussis The possible involvement of Tr1 induction is excluded.

  In the present specification, the inventors have shown that prior nasal administration of an attenuated strain of Bordetella pertussis (BPZE1), a causative microorganism of pertussis, in a Balb / c mouse model of severe interstitial pneumonia Reported to have provided effective and sustained protection against lethal attack by A virus and to a lesser extent against human H1N1 (A / PR / 8/34) influenza A virus. Although the cellular and molecular players involved in this cross-protection have not yet been identified, our data suggest that pertussis-specific adaptive immunity and Tr1-mediated down-regulation may not be involved Indicates. Importantly, the inventors have found that cross protection does not result in a reduction in viral load. Instead, protected BPZE1-treated mice exhibit minimal pulmonary immune symptoms, consistent with reduced neutrophil infiltration in their BALF and reduced production of various major pro-inflammatory cytokines and chemokines. Thus, our findings strongly suggest that protection against lethal interstitial pneumonia induced by influenza A virus can be achieved through attenuation of inflammation and suppression of cytokine storm, and pathogenic influenza A It demonstrates the potential use of BPZE1 bacteria as an effective preventive measure against protection against viral infections.

  All publications and patent applications cited herein are intended to be specifically and individually indicated as if each individual publication or patent application was incorporated by reference for all purposes. Are incorporated herein by reference in their entirety for all purposes.

  Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be made there without departing from the spirit or scope of the appended claims. This will be readily apparent to those skilled in the art in light of the teachings of the present invention.

reference

In one aspect, the present invention provides a method for inducing an immune response against an influenza virus in a mammal comprising administering a mutant Bordetella strain to the mammal, wherein the strain comprises a mutated pertussis toxin (ptx) gene, Methods are provided comprising a deleted or mutated skin necrosis (dnt) gene and a heterologous ampG gene. In some aspects, the Bordetella strain comprises a Bordetella pertussis strain. In some such aspects, the wild type Bordetella strain ampG gene has been replaced by the E. coli ampG gene. In other aspects, mutations in the ptx gene include substitution of amino acids involved in substrate binding and / or amino acids involved in catalysis. In some such aspects, substitutions of amino acids involved in substrate binding include R 9 K and substitutions of amino acids involved in catalysis include E129G. In some aspects, the Bordetella strain comprises a triple mutant strain. In some such aspects, the Bordetella strain comprises the BPZE1 strain. In other such aspects, the Bordetella strain is attenuated. In some aspects, the Bordetella strain comprises a live strain. In other aspects, the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene. In some aspects, the Bordetella strain does not include a heterologous expression platform for delivering a heterologous antigen to the mammalian respiratory mucosa. In other aspects, the method further comprises preventing or treating influenza infection in the mammal. In some aspects, the Bordetella strain is administered prior to influenza infection. In some such aspects, the Bordetella strain is administered about 6 weeks or prior to influenza infection. In other such aspects, the Bordetella strain is administered about 12 weeks or prior to influenza infection. In some aspects, the influenza virus comprises H3 or H1. In other aspects, the influenza virus comprises N2 or N1.

In another aspect, the invention provides a composition for treating or preventing influenza infection in a mammal, including a mutated Bordetella strain, wherein the strain is mutated pertussis toxin (ptx) gene, deleted or mutated Compositions comprising a skin necrosis (dnt) gene and a heterologous ampG gene are provided. In some aspects, the Bordetella strain comprises a Bordetella pertussis strain. In other aspects, the wild type Bordetella strain ampG gene is replaced with the E. coli ampG gene. In some aspects, mutations in the ptx gene include substitution of amino acids involved in substrate binding and / or amino acids involved in catalysis. In other aspects, substitutions of amino acids involved in substrate binding include R 9 K and substitutions of amino acids involved in catalysis include E129G. In some aspects, the Bordetella strain comprises a triple mutant strain.

In another aspect, the invention provides a Bordetella strain identified by Accession No. V09 / 009169.
[Invention 1001]
A method of inducing an immune response against an influenza virus in a mammal comprising administering a mutant Bordetella strain to the mammal, wherein the strain comprises a mutated pertussis toxin (ptx) gene, a deficiency A method comprising a lost or mutated skin necrosis (dnt) gene and a heterologous ampG gene.
[Invention 1002]
The method of the present invention 1001, wherein the Bordetella strain comprises a Bordetella pertussis strain.
[Invention 1003]
The method of the present invention 1002, wherein the wild-type Bordetella strain ampG gene is replaced by an E. coli ampG gene.
[Invention 1004]
The method of the present invention 1003, wherein the mutation in the ptx gene comprises a substitution of an amino acid involved in substrate binding and / or an amino acid involved in catalysis.
[Invention 1005]
The method of 1004 of this invention, wherein the substitution of amino acids involved in substrate binding comprises R 9 K and the substitution of amino acids involved in catalysis comprises E129G.
[Invention 1006]
The method of the present invention 1001, wherein the Bordetella strain comprises a triple mutant strain.
[Invention 1007]
The method of the present invention 1006, wherein the Bordetella strain comprises the BPZE1 strain.
[Invention 1008]
The method of the present invention 1001, wherein the Bordetella strain is attenuated.
[Invention 1009]
The method of 1001 of the present invention, wherein the Bordetella strain comprises a live strain.
[Invention 1010]
The method of the present invention 1001, wherein the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene.
[Invention 1011]
The method of the present invention 1001, wherein the Bordetella strain does not include a xenogeneic expression platform for delivering xenoantigens to the mammalian respiratory mucosa.
[Invention 1012]
The method of the present invention 1001, further comprising the step of preventing or treating influenza infection in a mammal.
[Invention 1013]
The method of the present invention 1001, wherein the Bordetella strain is administered prior to influenza infection.
[Invention 1014]
The method of the present invention 1013 wherein the Bordetella strain is administered about 6 weeks or prior to influenza infection.
[Invention 1015]
The method of the present invention 1014 wherein the Bordetella strain is administered about 12 weeks or prior to influenza infection.
[Invention 1016]
The method of 1001 of this invention, wherein the influenza virus comprises H3 or H1.
[Invention 1017]
The method of the present invention 1001, wherein the influenza virus comprises N2 or N1.
[Invention 1018]
The method of the present invention 1001, wherein the influenza virus comprises H3 and N2.
[Invention 1019]
The method of the present invention 1001, wherein the influenza virus comprises H1 and N1.
[Invention 1020]
The method of 1001 of this invention wherein the immune response comprises a Th1 immune response.
[Invention 1021]
The invention wherein the strain is administered to a mammal by subcutaneous (sc), intradermal (id), intramuscular (im), intravenous (iv), oral, or intranasal administration; or by infusion or by inhalation 1001 way.
[Invention 1022]
The method of the present invention 1001, wherein the strain is administered intranasally.
[Invention 1023]
The method of 1001 of the present invention, wherein the strain is administered to a mammal in need of protective immunity against influenza infection.
[Invention 1024]
The method of the present invention 1001, wherein the mammal is a child.
[Invention 1025]
The method of 1001 of the present invention, wherein the strain is administered once.
[Invention 1026]
The method of 1001 of this invention, wherein the strain is administered more than once.
[Invention 1027]
The method of the present invention 1026 wherein the strain is administered twice.
[Invention 1028]
The method of the present invention 1027, which is administered twice, about 3 weeks apart.
[Invention 1029]
The method of the present invention 1018 wherein the level of protection against influenza infection is greater than about 60%.
[Invention 1030]
The method of the present invention 1019, wherein the level of protection against influenza infection is greater than about 50%.
[Invention 1031]
The method of the present invention 1001 wherein the mammal is a human.
[Invention 1032]
A method of inducing a protective immune response against a H3N2 influenza virus in a human comprising the step of intranasally administering to said human a live attenuated BPZE1 strain prior to infection of the human with the H3N2 influenza virus, wherein the strain comprises A method that does not include a heterologous expression platform for delivering a heterologous antigen to the human respiratory mucosa.
[Invention 1033]
A method of inducing an immune response against an influenza virus in a human, the method comprising administering a live Bordetella strain to the human, wherein the strain carries a heterologous antigen to the human respiratory mucosa Not including the way.
[Invention 1034]
A method for protecting a mammal against diseases caused by influenza infection, comprising the step of administering to the mammal a mutant Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene Including.
[Invention 1035]
A method of providing protective immunity against influenza infection comprising administering to a mammal a mutant Bordetella strain comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene.
[Invention 1036]
A composition for treating or preventing influenza infection in a mammal comprising a mutated Bordetella strain comprising a mutated pertussis toxin (ptx) gene, a deleted or mutated skin necrosis (dnt) gene, and a heterologous ampG gene ,Composition.
[Invention 1037]
The composition of the invention 1036 wherein the Bordetella strain comprises a Bordetella pertussis strain.
[Invention 1038]
The composition of the invention 1037 wherein the wild type Bordetella strain ampG gene is replaced by the E. coli ampG gene.
[Invention 1039]
The composition of the invention 1038, wherein a mutation in the ptx gene comprises a substitution of an amino acid involved in substrate binding and / or an amino acid involved in catalysis.
[Invention 1040]
The composition of the invention 1039 wherein the substitution of amino acids involved in substrate binding comprises R 9 K and the substitution of amino acids involved in catalysis comprises E129G.
[Invention 1041]
The composition of the invention 1036 wherein the Bordetella strain comprises a triple mutant strain.
[Invention 1042]
The composition of the present invention 1041 wherein the Bordetella strain comprises the BPZE1 strain.
[Invention 1043]
The composition of this invention 1036 wherein the Bordetella strain is attenuated.
[Invention 1044]
The composition of the present invention 1036, wherein the Bordetella strain comprises a live strain.
[Invention 1045]
The composition of the present invention 1036, wherein the Bordetella strain does not contain a heterologous gene other than the heterologous ampG gene.
[Invention 1046]
The composition of the invention 1036 wherein the Bordetella strain does not comprise a xenogeneic expression platform for delivering xenoantigens to the mammalian respiratory mucosa.
[Invention 1047]
The composition of this invention 1036 further comprising a pharmaceutically suitable excipient, vehicle and / or carrier.
[Invention 1048]
The composition of this invention 1036 further comprising an adjuvant.
[Invention 1049]
The composition of this invention 1036 further comprising a small molecule capable of affecting an influenza infection.
[Invention 1050]
A composition comprising a Bordetella strain identified by accession number CNCM I-3585.
[Invention 1051]
A composition comprising a Bordetella strain identified by Accession No. V09 / 009169.
[Invention 1052]
A vaccine comprising the composition of the invention 1036 for treating or preventing influenza infection in a mammal.
[Invention 1053]
The vaccine of the present invention 1052 formulated for intranasal administration.
[Invention 1054]
A vaccine comprising the composition of the invention 1050 for treating or preventing influenza infection in a mammal.
[Invention 1055]
The vaccine of this invention 1054 formulated for intranasal administration.
[Invention 1056]
A vaccine comprising the composition of the present invention 1051 for treating or preventing influenza infection in a mammal.
[Invention 1057]
The vaccine of the present invention 1056 formulated for intranasal administration.

PTX is a major virulence factor responsible for the systemic effects of Bordetella pertussis infection and is one of the major protective antigens. Because of its nature, the natural ptx gene is such that the enzymatically active component S1 encodes an enzymatically inactive toxin, but so that the immunogenic nature of pertussis toxin is not affected. It can be replaced with a mutant form. This can be achieved by replacing arginine (Arg) at position 9 of the sequence with lysine (Lys) ( R 9 K ). Furthermore, glutamic acid (Glu) at position 129 can be replaced with glycine (Gly) (E129G). In general, each of these amino acid positions is involved in substrate binding and catalysis. In other aspects, other mutations may also be made, such as the mutations described in US Pat. No. 6,713,072, incorporated herein by reference, and any known or other mutation that can reduce toxin activity. . In one aspect, allelic exchange can be used first to remove the ptx operon, and then the variant can be inserted.

Claims (35)

  A pharmaceutical composition for inducing an immune response against an influenza virus in a mammal comprising a mutant Bordetella strain and a pharmaceutically acceptable material, wherein the strain is a mutated pertussis toxin (ptx) gene, A pharmaceutical composition comprising a deleted or mutated skin necrosis (dnt) gene and a heterologous ampG gene and no heterologous gene other than the heterologous ampG gene.   2. The pharmaceutical composition of claim 1, wherein the Bordetella strain comprises a Bordetella pertussis strain.   The pharmaceutical composition according to claim 2, wherein the wild-type Bordetella strain ampG gene is replaced by an E. coli ampG gene.   4. The pharmaceutical composition according to claim 3, wherein the mutation of the ptx gene comprises substitution of amino acids involved in substrate binding and / or amino acids involved in catalysis.   5. The pharmaceutical composition of claim 4, wherein the substitution of amino acids involved in substrate binding comprises K9R and the substitution of amino acids involved in catalysis comprises E129G.   2. The pharmaceutical composition of claim 1, wherein the Bordetella strain comprises a triple mutant strain.   7. The pharmaceutical composition according to claim 6, wherein the Bordetella strain comprises BPZE1 strain.   2. The pharmaceutical composition of claim 1, wherein the Bordetella strain is attenuated.   2. The pharmaceutical composition according to claim 1, wherein the Bordetella strain comprises a live strain.   2. The pharmaceutical composition according to claim 1, for preventing or treating influenza infection in a mammal.   The pharmaceutical composition according to claim 1, which is administered before influenza infection.   12. The pharmaceutical composition of claim 11, wherein the composition is administered about 6 weeks or before influenza infection.   13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered about 12 weeks or prior to influenza infection.   2. The pharmaceutical composition of claim 1, wherein the influenza virus comprises H3 or H1.   2. The pharmaceutical composition of claim 1, wherein the influenza virus comprises N2 or N1.   The pharmaceutical composition according to claim 1, wherein the influenza virus comprises H3 and N2.   The pharmaceutical composition according to claim 1, wherein the influenza virus comprises H1 and N1.   2. The pharmaceutical composition of claim 1, wherein the immune response comprises a Th1 immune response.   2. The mammal of claim 1 administered by subcutaneous (sc), intradermal (id), intramuscular (im), intravenous (iv), oral, or intranasal administration; or by infusion or by inhalation. Pharmaceutical composition.   2. The pharmaceutical composition of claim 1, wherein the composition is administered intranasally.   2. The pharmaceutical composition according to claim 1, which is administered to a mammal in need of protective immunity against influenza infection.   2. The pharmaceutical composition of claim 1, wherein the mammal is a child.   2. The pharmaceutical composition according to claim 1, which is administered once.   2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered more than once.   25. The pharmaceutical composition of claim 24, administered twice.   26. The pharmaceutical composition of claim 25, wherein the composition is administered twice, about 3 weeks apart.   17. The pharmaceutical composition of claim 16, wherein the level of protection against influenza infection is greater than about 60%.   18. The pharmaceutical composition of claim 17, wherein the level of protection against influenza infection is greater than about 50%.   2. The pharmaceutical composition according to claim 1, wherein the mammal is a human.   A pharmaceutical composition for eliciting a protective immune response against H3N2 influenza virus in a human comprising a live attenuated BPZE1 strain and a pharmaceutically acceptable material, wherein the composition comprises A pharmaceutical composition, wherein the strain is administered intranasally to the human prior to infection and the strain does not contain a heterologous gene other than the heterologous ampG gene.   A pharmaceutical composition for eliciting an immune response against influenza virus in humans, comprising a live mutated Bordetella strain and a pharmaceutically acceptable material, wherein the strain is a mutated pertussis toxin (ptx) gene, deletion Or a pharmaceutical composition comprising a mutated skin necrosis (dnt) gene and a heterologous ampG gene and no heterologous gene other than the heterologous ampG gene.   A pharmaceutical composition for protecting mammals against diseases caused by influenza infection, comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene A pharmaceutical composition comprising a mutant Bordetella strain and a pharmaceutically acceptable material, which does not contain a gene.   A pharmaceutical composition for providing protective immunity against influenza infection, comprising a mutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampG gene, and no heterologous gene other than the heterologous ampG gene, A pharmaceutical composition comprising a mutant Bordetella strain and a pharmaceutically acceptable material.   A pharmaceutical composition for treating or preventing influenza infection in a mammal comprising a mutated pertussis toxin (ptx) gene, a deleted or mutated skin necrosis (dnt) gene, and a heterologous ampG gene A pharmaceutical composition comprising a mutant Bordetella strain and a pharmaceutically acceptable material that does not contain a heterologous gene other than a gene.   35. A pharmaceutical composition according to claim 34 for use as a vaccine.

Images